Mantzoukas attended Swampscott High School, graduating in 1991. While attending, he was named captain of Swampscott’s boys’ soccer and track teams and was also president of the marching band. He then attended Middlebury College in Middlebury, Vermont; majoring in religion. He attempted to defend a “terribly written” honors thesis on religious iconography (which he admits to starting a week prior to his defense date) but was not given credit to graduate with honors.

After graduating college in 1995, Mantzoukas was granted a Watson Fellowship, traveling throughout North Africa and the Middle East studying religious and transcendental music for nearly two years. He has admitted to having a “horrible [grant proposal] but was charming in the room; so [he earned the grant].” At the time he left for Morocco for the Watson Fellowship, he had already had five or six auditions for the Blue Man Group, but he ultimately decided to travel before the audition process was complete. He lived in Morocco for about 7½ months while studying and recording Gnawa music. While abroad, he also traveled to Egypt, Israel, Turkey, Jordan, and Syria. During his time abroad, he was arrested numerous times; once in Morocco for having an expired tourist visa and again in Turkey to prevent him from traveling through an active war zone.

Soon after moving to New York City in 1998, he began performing comedy regularly at the Upright Citizens Brigade Theater (UCB) and was taught by Amy Poehler. He was a member of the improv team “Mother,” one of the UCB’s earliest house teams. During his time at UCB, Mantzoukas worked in the computer graphics department at J.P. Morgan for over three years to “make ends meet.”

Mantzoukas and comedian Ed Herbstman performed as the comedy duo, “The Mantzoukas Brothers,” for a number of years and were named the “Best Improv Duo” by Time Out New York magazine in 2006. Mantzoukas continued to write and perform as a duo with Jessica St. Clair appearing in the sketch show, “We Used to Go Out” and long-form improv show, “First Date.” Together, he and St. Clair pitched a pilot to HBO (which was ultimately not picked up) and also earned a deal with Comedy Central. The two were often compared to the likes of Nichols and May and continue to collaborate and perform on the podcast, Womp It Up!. Mantzoukas would later go on to teach advanced improv classes at UCB.

In 2008, NBC ordered a pilot for a comedy series written by Mantzoukas called Off Duty. The pilot was filmed in New York starring Bradley Whitford and Romany Malco but the series was not picked up. In interviews in 2014, Mantzoukas announced that he was working on a “loosely autobiographical” television show for Showtime about life after ending a long-term relationship.

In 2018, he replaced T.J. Miller as the voice of Mr. Mucus for the Mucinex commercials.