A native of Cincinnati, Ohio, Middleton appeared in many television programs in the 1950s and 1960s, including the CBS anthology series Appointment with Adventure. He played a dishonest candidate for the United States House of Representatives in an episode of ABC's The Real McCoys, starring Walter Brennan and Richard Crenna. In the storyline, Middleton falsely claimed to have previously been a farmer in a bid for the farm vote. Middleton was cast as "The Tichborne Claimant" in the NBC anthology series The Joseph Cotten Show. He was cast as Arthur Sutro in the 1961 episode "The Road to Jericho" of the ABC western series, The Rebel, starring Nick Adams.

One of his early works was as the narrator of the educational film "Duck and Cover". After appearing on the Broadway stage and live television, Middleton began appearing in films in 1954. He is also remembered on television as the boss Mr. Marshall on CBS's The Jackie Gleason Show and in film opposite Humphrey Bogart in The Desperate Hours (1955), Danny Kaye in The Court Jester (1955), Gary Cooper in Friendly Persuasion (1956), Richard Egan and Elvis Presley in Love Me Tender (1956), Dorothy Malone and Robert Stack in The Tarnished Angels (1958), Robert Taylor and Richard Widmark in The Law and Jake Wade (1958), and Dean Martin in Career (1959).

Among his several appearances in the long-running Alfred Hitchcock Presents, he portrayed a gangster in high places, Mr. Koster, in the 1956 episode "The Better Bargain". In 1958, he played the villain in the first episode of NBC's Bat Masterson western series, starring Gene Barry in the title role. He appeared in four episodes of The Untouchables, including the 2 part episode, "The Unhired Assassin", as Chicago mayor Anton Cermak. In 1961, he appeared in the episode "Accidental Tourist" on the James Whitmore ABC legal drama The Law and Mr. Jones. That same year, he portrayed the highly sympathetic but fiercely dedicated state executioner in an episode of Thriller (U.S. TV series) entitled "Guillotine". He also appeared in at least one episode of Bonanza (1964). Middleton appeared as defendant "Judge Daniel Redmond" in the 1963 Perry Mason episode, "The Case of the Witless Witness". In the early 1950s, Middleton appeared on Broadway in Ondine. Other significant film roles included The Court Jester (1955) as a grim and determined knight who jousts with Danny Kaye in the famous "pellet with the poison" sequence, and as Edwin M. Stanton in The Lincoln Conspiracy (1977). In between, he played an array of brutish mountain men, corrupt cigar-biting town bosses and lynch mob Leaders. Occasionally he showed some levity, as in his recurring role as Jackie Gleason's boss on The Honeymooners (1955) sketches. Middleton guest-starred on Get Smart as the KAOS villain "The Whip", intent on hypnotizing Agent 86 in the 1970 series finale "I Am Curiously Yellow".

In 1958, Middleton was cast in the episode "Ambush in Laredo" as Frank Davis, who attempts in Laredo, Texas, to merge five outlaw gangs into one, in the ABC Walt Disney miniseries Texas John Slaughter, with Tom Tryon in the title role.

Middleton was cast in ten episodes of the ABC family western drama, The Monroes, with costars Michael Anderson, Jr., and Barbara Hershey. In 1963 he portrayed Josh Green in the episode "Incident of the Mountain Man" on CBS's Rawhide.