McCallum began his career as a bit-part actor in British films of the 1950s. The Man from U.N.C.L.E., intended as a vehicle for Robert Vaughn, unexpectedly made McCallum into a sex symbol. Although he subsequently became a familiar face on television, he was never able to achieve the same level of popular success as he had done with his role as Kuryakin. His best-known roles were in Sapphire and Steel (opposite Joanna Lumley), as the lead in a 1970s remake of The Invisible Man, and as Judas Iscariot in The Greatest Story Ever Told.

He was married to the late actress Jill Ireland from 1957 to 1967. They had three sons, Paul, Jason (adopted), and Valentine.

McCallum co-starred with Charles Bronson in The Great Escape. He introduced Ireland to Bronson on the set and she later left him and married Bronson. McCallum married Katherine Carpenter in September 1967, and they have two children: Peter and Sophie.

He is currently starring in the CBS television series NCIS as Dr. Donald "Ducky" Mallard. In an inside joke on that program, when Agent Gibbs is asked the question, "What did Ducky look like when he was younger?," Gibbs simply responds, "Illya Kuryakin."

David McCallum recorded in the 1960s some albums for Capitol Records with producer David Axelrod, such as Music: A Bit More of Me (1966) and Music: It's Happening Now! (1967).

David McCallum appeared on stage in Australia when he appeared in the play Run For Your Wife , during (1987-1988), and the production toured the country. Other members of the cast in the production were Jack Smethurst, Eric Sykes and Katy Manning.