Mitroglou was born on 12 March 1988, in Kavala, Greece, and emigrated as an infant with his parents from his native country to Germany. He grew up in Neukirchen-Vluyn, North Rhine-Westphalia. He began his career at Maura Poulia. At age 11, he scored 24 goals in 16 matches. In 2001, he joined MSV Duisburg. After four seasons with the club, he joined Borussia Mönchengladbach. Mitroglou was a dominant player in the Under 19 Bundesliga. He scored 14 goals in 10 matches.

Mitroglou was first called up for the Greece national under-19 team in 2005. He scored a goal against the Netherlands in a 3–2 away win in 2007 UEFA European Under-19 Championship qualification. He also scored two goals in a 3–1 home loss against Germany in the same campaign. Mitroglou also scored in a 2–0 away win against Italy, two goals in a 2–2 away draw against Croatia and a goal in a 4–0 home win against Sweden. He participated at the 2007 UEFA European Under-19 Championship, in which he scored the only goal against Portugal at Linzer Stadion in the group stage, as well as in a 1–1 draw against Austria at the Waldstadion. In the knock-out stage, he scored a goal against Germany in a 3–2 win at Vorwärts Stadium. Mitroglou finished as the top goalscorer of the competition with three goals.

During the final stages of the season, Mitroglou played an important part in helping Olympiacos claim their 36th league title, scoring the last-minute winning goal (2–1) in their match against AEL, this time assisted by Mirnes Šišić. His first goal in the Superleague came in a 4–0 home win against Levadiakos. He then scored two goals in the last league fixture against Iraklis to seal a 3–1 win. He also scored in a 2–2 away draw against Iraklis in the Greek Cup semi-finals, after an assist from Michalis Konstantinou. In the 2007 Greek Super Cup, he scored the only goal as Olympiacos defeated AEL 1–0.

In 2007, Mitroglou was promoted to the Greece under-21 team. He scored two goals in a 3–1 home win against Macedonia in the UEFA European Under-21 Championship qualifiers, and later a goal in a 1–1 home draw against England and the only goal against Lithuania as the campaign continued.

After Valverde left Olympiacos, Mitroglou saw increased playing time. He scored his first goal of the season against Sheriff Tiraspol in a 2–0 first leg away win in the UEFA Champions League play-offs, also scoring in the return leg (a 1–0 home win). Mitroglou scored the equalizing goal in an eventual 2–1 defeat of Standard Liège in a UEFA Champions League group stage match on 21 October 2009. He also scored in home wins against Asteras Tripoli (3–0) and Ergotelis (2–1). His next goal came in an away draw (2–2) against PAS Giannina after an assist by Dudu Cearense. He also scored a goal in a heavy 3–1 away loss to Panserraikos.

In 2009, Mitroglou was selected for the senior Greece side for the first time. He scored his first goal in a 3–2 away win over Norway on 15 August 2012. On 11 September 2012, he scored a goal in a 2014 FIFA World Cup qualifying match against Lithuania, which ended in a 2–0 victory. He went on to score both goals in a 2–0 away friendly win against Austria on 14 August 2013.

Ernesto Valverde returned as head coach of Olympiacos for the 2010–11 season. Despite being a starter and scoring a spectacular goal in the opening league match against Iraklis (1–2 away loss), Mitroglou only made four more appearances as Valverde chose to use Kevin Mirallas and Marko Pantelić instead. Mitroglou participated in the international match “8th Match Against Poverty” on 14 December 2010 (held at the Georgios Karaiskakis Stadium) as a player in the Olympiacos All-Star team and scored two goals.

In January 2011, Mitroglou joined Panionios on a six-month loan from Olympiacos. In his debut, he scored both goals in a historic 2–0 away win against Aris, the first for the club in 50 years. His next goals came at a 1–1 home draw against Panathinaikos and a 1–1 away draw against AEK Athens, the latter after an assist from Georgios Galitsios. He scored the winning goal (2–1) against Atromitos and converted a penalty at the 3–3 home draw against AEL. He added another one at a 1–1 away draw against Iraklis. He scored his last goal at the 2–1 away win against Kerkyra after an assist from Fanouris Goundoulakis. Mitroglou ended this year with nine goals in the Superleague, one with Olympiacos and eight with Panionios.

On the second matchday of the Superleague Greece, he provided an assist to David Fuster in a 2–1 home win against Atromitos. On 1 September 2013, he scored his first career hat-trick in a 5–0 away win against Levadiakos after three assists from Fuster. He scored his second hat-trick on the next fixture in a 4–0 home win against Skoda Xanthi, becoming the first player in Olympiacos history—as well as the Greek League generally—to score two consecutive hat-tricks, while also having also scored in an international match between these two fixtures.

Mitroglou scored the only goal in a victory over Liechtenstein, after an assist from Vasilis Torosidis, in their penultimate World Cup qualifying match on 6 September 2013. He scored two goals on 15 November in Greece’s 3–1 home win against Romania in the first leg of their 2014 FIFA World Cup qualifying play-off. Four days later, he scored the opening goal of the return leg in Romania, which ended in a 1–1 draw and sent Greece to the World Cup final stages in Brazil with a 4–2 aggregate victory.

On 27 November, Mitroglou suffered an injury in a 2–1 away loss against Paris Saint-Germain, keeping him on the sidelines for over a month. On 19 December, amid much transfer speculation, Mitroglou renewed his contract with Olympiacos until 2017. On 10 January 2014, he recovered to come on as a substitute against Atromitos.

On 31 January 2014, Mitroglou signed a four-and-a-half-year contract for a transfer fee believed in the region of £12 million with English Premier League club Fulham. On 22 February, he made his debut for Fulham in a 1–1 draw at West Bromwich Albion, replacing Hugo Rodallega after 61 minutes. Mitroglou was the highest transfer fee in Fulham’s history, and despite Fulham pinning their hopes of staying in the Premier League on the Greece international, they were relegated in May 2014. Due to various injuries and fitness problems, Mitroglou played in only two more Premier League matches before the end of the 2013–14 season, starting only one, away against Cardiff City.

On 31 August 2014, Mitroglou returned to Olympiacos on a season-long loan from Fulham. He made his debut on 13 September, replacing Dimitris Diamantakos in the 55th minute of a 3–0 home win over OFI Crete. Three days later, he scored what proved to be the game winner in a 3–2 win over Atlético Madrid in the UEFA Champions League group stage.

On 6 August 2015, Mitroglou joined Portuguese champions Benfica on loan until the end of the season. He made his debut three days later, replacing compatriot Andreas Samaris for the final 18 minutes of a 0–1 loss to rivals Sporting CP in the 2015 Supertaça Cândido de Oliveira at the Estádio Municipal de Aveiro. In their first match of the Primeira Liga season against Estoril, he opened the scoring in a 4–0 win at the Estádio da Luz. On 11 September, he scored in each half of a 6–0 win in the league against Lisbon neighbours Belenenses.

On 23 January 2016, Mitroglou scored with a backheel goal in a 3–1 win against Arouca, and on 3 February, he scored a hat-trick against Belenenses in a 0–5 away victory. It was his first hat-trick in the season, scoring for the fifth consecutive match in a row. On 13 February, he opened the score in a 2–1 home loss against rivals Porto, struggling for the win before goalkeeper Iker Casillas made a save at close quarters to stop him from scoring the equalizer. One week later, he scored for the seventh match in a row by opening the score in a 1–3 win at Paços de Ferreira; at the final whistle, a Benfica supporter entered the field and knelt before Mitroglou, and then tried to shine his boots with a scarf. On 5 March, Mitroglou scored the only goal in the Lisbon derby at Sporting CP’s Estádio José Alvalade, giving Benfica the lead in the league, ahead of their opponents. Later on, he scored a brace in a 5–1 home win against Braga in the Primeira Liga on 1 April, increasing his tally to 18 goals in the league, with 13 goals scored in 12 matches. He ended the league campaign with 20 goals in 32 matches, winning the Primeira Liga title on 15 May after a 4–1 home win over Nacional.

Two days after the league title celebrations, Benfica announced Mitroglou had signed a permanent contract with the club set to last until 2020, for a €7 million transfer fee, as disclosed by Football Leaks. On 18 January 2017, he scored a hat-trick in a 6–2 home win over Leixões in the Taça de Portugal quarter-finals. On 28 February 2017, Mitroglou scored twice against Estoril in the first leg of the Taça de Portugal semi-finals.

On 31 August 2017, French Ligue 1 club Marseille announced the signing of Mitroglou on a four-year contract for a transfer fee of €15 million to Benfica. On 28 September, Mitroglou made his competitive debut for Marseille in a 2017–18 UEFA Europa League group stage 1–0 away loss against Red Bull Salzburg, coming on as a substitute in the 79th minute for Maxime Lopez.

On 6 February 2018, Mitroglou scored his first hat-trick for Marseille—to go along with one assist,—in a 9–0 blowout win over Bourg-en-Bresse in the Coupe de France.

On 31 January 2019, Mitroglou was loaned out to Galatasaray in Süper Lig until 30 June 2020.

On 22 August 2019, PSV Eindhoven announced they would take on Mitroglou on a loan until the end of the 2019–20 Eredivisie season.