He has voiced Penn Zero in the Disney XD animated series Penn Zero: Part-Time Hero (2014–2017), Harold Hutchins in Captain Underpants: The First Epic Movie (2017), Sam Coleman in Godzilla: King of the Monsters (2019), and Terry Opposites in the Hulu adult animated sci-fi series Solar Opposites.

In 2007 a sketch video for Chicken McNuggets he made with Fernando Sosa in Chicago was purchased by McDonald's and used as a commercial.

[14] On May 31, 2011, Middleditch joined the cast of the Paramount Pictures' comedy film Fun Size, directed by Josh Schwartz.

[21] On January 30, 2013, Middleditch joined HBO's comedy pilot Silicon Valley playing Richard Hendricks.

[6] This was a result of a "random" audio sketch he once made for a stand-up routine which he would later animate himself and pitched to several people, including Mike Judge's production partners John Altschuler and Dave Krinsky, who would later create the show.

[24][25] In 2016, Middleditch earned his first Primetime Emmy Award nomination for Outstanding Lead Actor in a Comedy Series for his performance in the show.

[26] On April 23, 2013, he was added to the cast of the comedy film Search Party, playing Nardo, who travels to Mexico to win his fiancé back, starring alongside one of his Silicon Valley alumni, T.J.

[27] Middleditch made a small cameo appearance in the 2013 film The Wolf of Wall Street as a broker who is fired for cleaning his fish bowl during office hours.

On The Pete Holmes Show, he played Nightcrawler and Gambit from the X-Men and Vega and Ken from Street Fighter II.

[37] In October 2020, Middleditch began playing the lead role of Drew Dunbar in the CBS comedy series B Positive which was written by Chuck Lorre.

[38][39] The series was originally supposed to air in March 2020, but production was halted due to the COVID-19 pandemic; however, filming of the pilot had already taken place and so the show was moved to be CBS' only fall debut.