On 5 December, in a dramatic 1–1 away draw against Torino in domestic league action, Manolas brought down Andrea Belotti inside the area, whereupon Maxi López coolly converted the subsequent 94th-minute penalty kick. Despite replays suggesting the Greek defender may well have gotten the ball in the challenge, the referee nonetheless awarded the penalty. On 11 December, towards the end of a Champions League match against Belarusians BATE Borisov, Manolas engaged in a quarrel with teammate Miralem Pjanić for mishandling a ball possession. As reported by La Gazzetta dello Sport, the two players continued the fighting into the team's locker room. On 17 February 2016, in a 1st leg UEFA Champions League game for the last 16, he was the indisputable leader of the defence, despite the 2–0 home loss from the Spanish giants Real Madrid. Manolas closed down Karim Benzema quite well throughout the game. Most of the low crosses were taken care of well by him but he could do nothing about the two goals. On 20 April 2016, in a match for Serie A, Manolas scored his second goal in the season, giving an assist in a glorious home win against Torino F.C.
On 25 July 2016, coach Luciano Spalletti has insisted that the international defender is happy at A.S. Roma and will stay at the club, despite every summer speculations involving Manchester United, Arsenal and Chelsea's interest about the Greek defender. Arsenal were so displeased by the way Roma handled the talks that they later refused to allow Jack Wilshere to go there on loan. Manolas began the 2016–17 campaign as the de facto leader of the Roma defence.
On September 2016, A.S. Roma are obliged to pay the sum of €6.5 million to Olympiakos for the international defender. Roma did not want to sell the Greek defender this summer as Olympiakos had a 50% next percentage sale until this August. Roma also rejected a bid from Arsenal in the region of €39 million for Manolas and therefore the player stayed put. Now Olympiakos will receive from Roma the fee of €6,5 million and Roma will activate the clause obtaining the 100% of the player. Manolas is considered the best marker of 'calcio' and Barcelona have been following him for a while. The Greek international has always been on a list of defenders that Barcelona have been interested in. With Thomas Vermaelen's loan to Roma they received an option to buy on Manolas at the summer of 2017. On 2 October 2016, he scored the second goal sealing a 2–1 home win against rivals Inter, as well as made the Inter captain Mauro Icardi disappear. On 18 October 2016, Manolas rejected A.S. Roma's proposal on the extension of his contract, which expires in 2019, despite the fact that the management was ready to raise the salary of the Greek centre back from €1.8 million to €2.5 million per year. According to the newspapers sources, the 25-year-old international player saw that almost 12 players had a higher salary than his and now expects an improved offer.
On 23 October 2016, Manolas reached 100 appearances with the jersey of Roma, in a 4–1 home win Serie A game against Palermo. On 31 October, he broke his nose in AS Roma's 0–0 away draw against Empoli, in a game where the Greek defender simply covered every possible hole, thus missing Europa League clash against FK Austria Wien and Serie A match with Bologna at the Olimpico, the Giallorossi said. On 4 December 2016, in one of the most spectacular Roman derbies in history, Manolas showed the usual ability both in anticipating and blocking his opponents, giving Balde Diao Keita and Ciro Immobile a very difficult afternoon. On 13 December 2016, Roma's center-back trio had an amazing performance, especially Manolas and Federico Fazio who combined 15 clearances, 11 interceptions and won 12 duels. Manolas' impact surpassed just defensive duties as he assisted for Radja Nainggolan's 1–0 winner against A.C. Milan. On 18 December 2016, Manolas according to doctors' report, will be out for around two weeks after picking up another injury in Roma's 1–0 defeat to Juventus. Manolas was forced off in the latter stages at Juventus Stadium, and he will now miss Roma's match against Chievo, the defender having already been struck down with a broken nose and muscle strain this season. On 8 January 2017, he returned to the squad playing as a late substitute in a 1–0 away win against Genoa. On 25 May 2017, a week before the end of the season, Manolas and Diego Perotti came a step away from putting their hands on them during training. To avoid the worst, only the provisional intervention of teammates,along with the Luciano Spalletti's decision to send them in the dressing room, without even finishing the training, helped to lower the tension.