His first film, The Light Ahead (1939), directed by Henry Felt and Edgar G. Ulmer, is notable for being entirely in Yiddish. Opatoshu appeared as the homicide detective, Sgt. Ben Miller, in the film noir, The Naked City (1948) produced by Mark Hellinger. In 1958, he played a supporting character in The Brothers Karamazov with soon-to-be Star Trek co-star william Shatner. He also portrayed Herr Jacobi, one of the people who help Paul Newman and Julie Andrews escape from East Germany in Alfred Hitchcock's 1966 film Torn Curtain. He also played the father of Benny Rampell in 1963's "The Cardinal" un credited.

David Opatoshu was survived by his wife, Lillian Weinberg, a psychiatric social worker, whom he married on June 10, 1941. They had one child together, a son, Danny. Lillian died on May 13, 2000.

His career in television began in 1949 and lasted through the 1980s. In the fall of 1953, he played a theatrical agent representing Ezio Pinza's title character in the NBC situation comedy Bonino. Other costars were Mary Wickes, Chet Allen, and Van Dyke Parks. The series focused upon an Italian American opera singer trying to rear his six children after having been widowed.

He appeared on Broadway in Silk Stockings in 1956, The Wall in 1960, and Bravo Giovanni in 1962, and others.

In 1963 he co-starred with James Doohan in an episode of The Twilight Zone, titled "Valley of the Shadow". He guest-starred in the 1964 The Outer Limits episode "A Feasibility Study"; in the 1965 Voyage to the Bottom of the Sea episode "The Price of Doom'; and in the 1965 two-part episode of The Man from U.N.C.L.E. called "The Alexander the Greater Affair". In 1967 he played Anan 7 in the original Star Trek series episode "A Taste of Armageddon", and also in the 1969 season 3 Ironside episode "L'Chayim", and in Mannix, in the episode "A Pittance of Faith", as Mr. Lardelli, in the same year.

David Opatoshu also wrote the screenplay for the film Romance of a Horsethief (1971), based on a novel by his father, Joseph Opatoshu.

He played the Irgun leader (and Ari Ben Canaan's estranged uncle) in Otto Preminger's 1960 film Exodus. In 1967, Opatoshu played Morris Kolowitz, the father of the main character David (Reni Santoni), in Carl Reiner's directorial debut Enter Laughing. In the 1977 film, Raid on Entebbe, he played the part of Menachem Begin, a film based on the actual Operation Entebbe and the freeing of hostages at Entebbe Airport in Entebbe, Uganda on July 4, 1976. Ironically, he'd played Begin's fictional counterpart in Exodus.

In the next year, 1970, he played in Daniel Boone, season six, as Tamenund. In the "No Way to Treat a Relative" episode of the 1973 situation comedy Needles and Pins (never broadcast because of the show's cancellation), the Kojak episode "Both Sides of the Law", the 1977 The Bionic Woman episode "Doomsday is Tomorrow", the 1981 Buck Rogers in the 25th Century episode "Time of the Hawk", and the 1981 miniseries Masada. In 1986 he played an Iranian ambassador in the TV thriller Under Siege, about Islamic terrorist attacks in the United States. On October 30, 1989, Opatashu guest-starred as the Tenctonese ex-slave "Paul Revere", in the episode "Night of the Screams", of the television series Alien Nation. In 1991 he won an Emmy Award for his guest appearance in the episode "A Prayer for the Goldsteins" of the ABC series Gabriel's Fire.