Jacobi came to prominence for his role as Mr. Van Daan in the 1955 Broadway production of The Diary of Anne Frank which he reprised in the 1959 film version.

He also acted in the films Irma la Douce (1963), Little Murders (1971), Everything You Always Wanted to Know About Sex* (*But Were Afraid to Ask) (1972), Next Stop, Greenwich Village (1976), The Lucky Star (1980), Arthur (1981), My Favorite Year (1982), and Avalon (1990).

Other Broadway performances included Paddy Chayefsky’s The Tenth Man (1959), Woody Allen’s Don’t Drink the Water (1966), and Neil Simon’s debut play Come Blow Your Horn (1961), in which he portrayed the playboy protagonist’s disappointed father.

[4] Other notable films in which he appeared include Irma la Douce (1963), Penelope (1966), Everything You Always Wanted to Know About Sex* (*But Were Afraid to Ask) (1972) as Sam Musgrave, a middle-aged married man experimenting with women's clothes, Arthur (1981) as the lucky florist, My Favorite Year (1982) as Benjy's unsophisticated Uncle Morty, and in Amazon Women on the Moon (1987), as a man named Murray who got zapped into the television and is wandering throughout sketches looking for his wife.

In Barry Levinson's Avalon (1990), in a semi-dramatic role, as one of four Russian brothers (elders) trying to build a future in Baltimore in the early 20th century, with the memorable comic relief catchphrase, "You cut the turkey!?"

In the summer of 1976, Jacobi was the star of a CBS comedy series Ivan the Terrible, in which he played a Russian headwaiter living with nine other people in a small Moscow apartment.