Among his most famous roles in a long career are those of Gerald O'Hara in Gone with the Wind, Doc Boone in Stagecoach, Uncle Billy in It's a Wonderful Life, Pat Garrett in The Outlaw, and Mayor Jonas Henderson in High Noon.

Considered one of the finest character actors in film,[6] in 1939 alone he had key roles in Stagecoach, Mr. Smith Goes to Washington, Only Angels Have Wings, The Hunchback of Notre Dame, and Gone with the Wind.

Throughout the 1940s and 1950s, Mitchell acted in a wide variety of roles in productions such as 1940's Swiss Family Robinson, 1942's Moontide, 1944's The Keys of the Kingdom (as an atheist doctor) and High Noon (1952) as the town mayor.

He is probably best known to audiences today for his role as sad sack Uncle Billy in Capra's Christmas classic It's a Wonderful Life (1946) with James Stewart.From the 1950s and into the early 1960s, Mitchell worked primarily in television, appearing in a variety of roles in some of the most well-regarded early series of the era, including Playhouse 90, Dick Powell's Zane Grey Theater (in a pilot episode that became the CBS series Johnny Ringo), and Hallmark Hall of Fame productions.

In 1955, he played Kris Kringle in The 20th Century-Fox Hour version of The Miracle on 34th Street opposite Teresa Wright and MacDonald Carey.

Mitchell's last role was on the stage, portraying Columbo, a detective character previously played by Bert Freed on an episode of The Chevy Mystery Show and later made famous on NBC and ABC television by Peter Falk.

[7] As a part of a 2023 cataloging project of the cremains in the vaults, Mitchell's family confirmed his wishes that his ashes remain in vaultage and not be made publicly accessible.