During his Hollywood career and in his 1984 autobiography Things I Did and Things I Think I Did, Negulesco claimed to have been born on 29 February 1900; he was apparently motivated to make this statement because birthdays on Leap Year Day are comparatively rare (and even though 1900 was not a Leap Year in the Gregorian calendar, it was under the Julian calendar, which applied in Romania at that time).

Born in Craiova, he attended Carol I High School. In 1915 he moved to Vienna, and then went to Bucharest in 1919, where he worked as a Painter before becoming a stage decorator in Paris. In 1927, he visited New York City for an exhibition of his paintings and settled there. He then made his way to California, at first working as a portraitist.

In 1934, he entered the film industry, first as a Sketch Artist, then as an assistant Producer, second unit Director. In the late 1930s, he became a Director and Screenwriter. He made his reputation at Warner Brothers by directing short subjects, particularly a series of band shorts featuring unusual camera angles and dramatic use of shadows and silhouettes.

Negulesco's first feature film as Director was Singapore Woman (1941). In 1948 he was nominated for an Academy Award for Directing for Johnny Belinda. In 1955 he got a nomination the BAFTA Award for Best Film for How to Marry a Millionaire. His 1959 movie, The Best of Everything, was on Entertainment Weekly's Top 50 Cult Films of All-Time list.

From the late 1960s he lived in Marbella, Spain, where he died, at age 93, of heart failure.