He also had a recurring guest role as a doctor on the animated Adult Swim show Tom Goes to the Mayor and appeared in several episodes of Tim and Eric Awesome Show, Great Job! in a recurring role as Tairy Greene.

In 2006, Galifianakis was featured in Fiona Apple's music video for the song "Not About Love" where he is seen lip-synching the lyrics to the song. A year later Kanye West employed Galifianakis and indie rock musician Will Oldham for similar purposes in the second version of the video for his song "Can't Tell Me Nothing". In June 2006 Galifianakis released the single "Come Over and Get It (Up in 'Dem Guts)", a comedic rap/hip-hop/dance song which features Apple's vocals.

Galifianakis was, together with Patton Oswalt, Brian Posehn, and Maria Bamford, one of the four Comedians of Comedy, a periodic packaged comedy tour in the style of The Original Kings of Comedy and the Blue Collar Comedy Tour. They chose to perform at live rock clubs as opposed to comedy clubs to try to reach a different audience. Much of the tour was taped, and has been featured in both a short-lived TV series on Comedy Central and a full length movie that has appeared at SXSW and on Showtime.
On February 22, 2008, he made an appearance on the Jackassworld.com: 24 Hour Takeover. He interviewed various members of the Jackass cast.

Galifianakis starred in the independent film Visioneers which played in select cities in 2008. This was Galifianakis's first starring role in a film and was given a direct-to-DVD release. That same year, Galifianakis appeared in a web video series of advertisements for Absolut vodka, along with Tim Heidecker and Eric Wareheim, creating a parody of the Golden Girls in which one has a deep anger issue, breaking the fourth wall in exasperation and outright violence on the set. He also completed the pilot Speed Freaks for Comedy Central.

Galifianakis has a series of videos on the Funny or Die website titled "Between Two Ferns With Zach Galifianakis" where he conducts interviews with popular celebrities between two potted ferns. He has interviewed Jimmy Kimmel, Michael Cera, Jon Hamm, Natalie Portman,Charlize Theron, Bradley Cooper (with a brief appearance by Carrot Top), Conan O'Brien and Andy Richter (with a brief appearance by Andy Dick), Ben Stiller, Steve Carell, Sean Penn, Bruce Willis, Tila Tequila, Jennifer Aniston and Will Ferrell. His interview style consists of typical interview questions, bizarre non sequiturs and sometimes inappropriate sexual questions and comments.

Galifianakis co-starred in the comedy The Hangover and earned the MTV Movie Award for the Best Comedic Performance. Following The Hangover's release, Galifianakis was prominently advertised in subsequent films that featured him in supporting roles. These included G-Force, Youth in Revolt, and the Oscar-nominated film Up in the Air.

Galifianakis is currently a member of the regular cast playing a supporting role in the HBO series Bored to Death. He hosted Saturday Night Live on March 6, 2010 during the show's 35th season, during which he shaved his beard mid-show for a sketch, and then closed the show wearing a fake beard. He hosted again on March 12, 2011 and shaved his head this time, in a Mr. T-like hairstyle, which was allegedly supposed to be used for a sketch that never aired due to time constraints. In 2010, he starred in several films, including Dinner for Schmucks, It's Kind of a Funny Story, and Due Date.

On October 29, 2010, while debating marijuana legalization on the show HBO's Real Time with Bill Maher, Galifianakis appeared to have smoked marijuana on live television; host Bill Maher denied that it was real marijuana in an interview with Wolf Blitzer during an episode of The Situation Room.
In 2011, he returned as Alan Garner in the sequel, The Hangover Part II, which was set in Thailand.