In the early 1980s, Wilson moved to Los Angeles to pursue his career. He shared an apartment with fellow aspiring comedians Andrew Dice Clay and Yakov Smirnoff, and later joked that he "taught them both about America."

His breakthrough role was the bully Biff Tannen in the 1985 film Back to the Future. He returned in the sequels Back to the Future Part II and Part III to reprise not only his role as Biff, but also to play Biff's grandson Griff Tannen and great-grandfather Buford "Mad Dog" Tannen. In every Back to the Future film, he ends up in a pile of manure (in reality, a heap of decayed sphagnum and other plant matters) after trying to kill or hurt Michael J. Fox's character Marty McFly. He reprised his role as Biff and voiced various Tannen relatives in the animated series. Wilson did not reprise his role as Biff in the initial versions of Telltale's Back to the Future: The Game released in 2011, being replaced by Kid Beyond. When the game was ported to the PlayStation 4, Xbox 360 and Xbox One in 2015 in commemoration of the original film's 30th anniversary, Wilson returned to provide Biff's voice in these newer versions.

Wilson played the role of a Detroit police officer in the 1988 film Action Jackson.

In 1992, he voiced gangster Tony Zucco in Batman: The Animated Series and police detective Matt Bluestone in the animated series Gargoyles. He later went to co-star with Mark Hamill in Wing Commander III: Heart of the Tiger, a video game. It was the third chapter in the Wing Commander series, but the first to feature live action and was extremely popular at the time. The character played by Wilson was Major Todd "Maniac" Marshall, a fellow starfighter pilot of Hamill's character. Wilson also starred in the sequels Wing Commander IV: The Price of Freedom (1995) and Wing Commander: Prophecy (1997) and contributed his voice to the animated series Wing Commander Academy (1996) in the same role. He also guest starred in an episode of Lois and Clark: The New Adventures of Superman in 1997.

Wilson also played Simon, Hilda's dream date made out of dough, in Sabrina the Teenage Witch in 1996.

Wilson played McKinley High School's Coach Ben Fredricks in the 1999–2000 NBC comedy-drama Freaks and Geeks. Coach Fredricks dated Bill Haverchuck's mother. Wilson was briefly reunited with his Back to the Future co-star Christopher Lloyd in the 1994 film Camp Nowhere.

Wilson practices Catholicism, and released a Contemporary Christian album in 2000 called In the Name of the Father.

In 2003, Wilson appeared on the screen in a mockumentary movie called Trial and Error: The Making of Sequestered. Wilson has often lent his voice to animated productions such as Batman: The Animated Series (1993), The New Batman Adventures (1998), Max Steel (2000), Disney's Atlantis: Milo's Return (2003), and The SpongeBob SquarePants Movie (2004). As well as the animated roles, he has continued to voice characters in computer and video games. He had supporting roles in the film Larry the Cable Guy: Health Inspector and the TV drama Ghost Whisperer.

In 2004, Wilson played the role of Noah Curry in the Pasadena Playhouse production of the musical 110 in the Shade, also starring Marin Mazzie and Jason Danieley. In 2005, Wilson released his comedy album, Tom Wilson Is Funny!.

Wilson is a Painter. Many of his paintings focus on classic children's toys. In 2006, he was selected to join the California Featured Artist Series at Disneyland.

In 2007, Wilson appeared in the episode "Whatever It Takes" of the drama series House M.D. as Lou, the Father of Dr. House's patient.

In 2008, Wilson appeared in the episode "The Baby in the Bough" of another Fox drama, Bones. He played Chip Barnett, owner of a tire recycling plant.

In March 2009, he appeared as himself, busking, in Vidiotic, a comedy pilot on the British channel BBC Three.

He has hosted a podcast, Big Pop Fun, on the Nerdist Network starting in November 2011. The podcast features Tom sharing stories of his career, as well as informal chats with show Business friends including Samm Levine, Blake Clark, Steve Oedekerk, "Weird Al" Yankovic, and more.