He appeared in the films The Dictator, The Long Dumb Road, Sleeping with Other People, They Came Together, Conception, and John Wick: Chapter 3 – Parabellum.

He has had recurring roles on three TV series created by Michael Schur: Parks and Recreation (as Dennis Feinstein), Brooklyn Nine-Nine (as Adrian Pimento), and The Good Place (as Derek Hofstetler).

[6] In interviews, he has cited Stewart Copeland, Stephen Perkins from Jane's Addiction, and Jimmy Chamberlin from The Smashing Pumpkins as his biggest influences as a young musician.

[13] After graduating from college in 1995, Mantzoukas was granted a Watson Fellowship and traveled throughout North Africa and the Middle East studying religious and transcendental music for nearly two years.

[9] 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.

[18][19][20] During his time at UCB, Mantzoukas worked in the computer graphics department at J.P. Morgan for over three years to "make ends meet.

[26] Mantzoukas has appeared in films such as Baby Mama, I Hate Valentine's Day, The Dictator, and John Wick: Chapter 3 – Parabellum, and starred in The Long Dumb Road.

[9][30] Mantzoukas has done extensive voice acting work, including main roles on the animated series Big Mouth, Close Enough, HouseBroken, and Star Trek: Prodigy.

[32] In January 2025, Mantzoukas was announced as one of the contestants for the 19th series of Taskmaster, against Fatiha El-Ghorri, Mathew Baynton, Rosie Ramsey, and Stevie Martin.

[9] 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.

[9] Mantzoukas earned credit for writing the screenplay draft of the buddy cop comedy Ride Along, starring Ice Cube and Kevin Hart.