After nightclub performing and stage work, Fowley appeared in 1933 in his first film, The Mad Game, alongside Spencer Tracy. Early in his acting career, he was usually cast as movie heavies or Gangsters in B-movies, including Charlie Chan and Laurel and Hardy features.

In the 1950s, he appeared as himself on NBC's The Donald O'Connor Show. In 1954, he demonstrated his comic appeal when he appeared alongside Gracie Allen in The George Burns and Gracie Allen Show. He was cast in 1956 as Bob Egan in the "Two-Fisted Saint" episode of the religious anthology series Crossroads. He portrayed a con man in two episodes of the NBC sitcom It's a Great Life. He also guest-starred on Reed Hadley's CBS legal drama The Public Defender. He appeared, too, on the ABC situation comedy The Pride of the Family and on the NBC western series The Californians and Jefferson Drum. He was cast on two Rod Cameron series, the syndicated City Detective and the western-themed State Trooper, and in John Bromfield's series, U.S. Marshal. He guest-starred too in the David Janssen crime drama Richard Diamond, Private Detective.

For several seasons, Fowley played the key supporting role of John H. "Doc" Holliday in the 1955-1961 western television series The Life and Legend of Wyatt Earp after having appeared as Doc Fabrique in the show's premiere season. This role allowed Fowley to demonstrate his flair for comedy and other acting skills as a clever, sharp-witted, sardonic, cynical, alcoholic, poker-playing foil to the square-jawed, milk-drinking, church-going Wyatt Earp (Hugh O'Brian), whom Holiday nicknamed "Deacon" due to his rigid sense of morality. Not at all so encumbered Doc would occasionally take the law into his own hands behind Earp's back to protect his friend from legal action or even death when the marshal was legally or morally ham-strung. Holliday, as played by Fowley, having no Problem working around morals or the law, could be either hilarious or cold-blooded.

He had a role in the syndicated 1959-1960 western Pony Express in the episode "Showdown at Thirty Mile Ridge". He was cast in 1963 in Miracle of the White Stallions.

In 1964, Fowley made a guest appearance on the CBS courtroom drama series Perry Mason playing agent Rubin Cason in "The Case of the Bountiful Beauty."

In 1965, he was cast as "Sorrowful" in episode 83 of the series "The Virginian".

In 1966, he appeared as "Rufus C. Hoops" in "The Search" season 2, episode 24, of the series "Daniel Boone". Original air date for this episode was March 3, 1966.

In 1967, Fowley guest-starred on the short-lived CBS western Dundee and the Culhane with John Mills.

In 1968, he appeared in episode 273 of My Three Sons as an old pal of Uncle Charley.

Fowley continued to act into the 1970s and was frequently billed as "Douglas V. Fowley". One of his last roles was as Delaney Rafferty in Disney's The North Avenue Irregulars, in which he dressed in drag.

Fowley portrayed retired businessman Robert Redford in Detective School (1979).

Fowley died nine days before what would have been his 87th birthday. He was buried at the Murrieta, California, Laurel Cemetery.