Benaderet first received notice for her radio work in the 1940s playing Millicent Carstairs on Fibber McGee & Molly, telephone operator Gertrude Gearshift on The Jack Benny Program, and appeared on several Amos 'n Andy radio shows, usually as a store clerk trying to assist Andy and Kingfish in a purchase. Benaderet also played Blanche Morton, next door neighbor to George Burns and Gracie Allen, on both radio and television.

She played Lucille Ball's best friend, Iris Atterbury, on the 1940s radio series My Favorite Husband. When Ball and husband Desi Arnaz decided to create a very similar television series called I Love Lucy, Benaderet was first choice to fill the role of Ethel Mertz but was unavailable to take the role since she had already signed for Burns and Allen's television adaption of the radio program. Vivian Vance, an almost unknown character actress and singer, was eventually cast in the part. Benaderet did get to guest on I Love Lucy in a very amusing appearance as Miss Loomis, a love-starved spinster neighbor.

Benaderet was also seriously considered for the role of Granny in The Beverly Hillbillies, created by her producer from The Burns & Allen Show, Paul Henning, who ultimately felt she was too buxom and womanly for the role he envisioned as a frail but caustic little spitfire. Irene Ryan was eventually cast. Henning did cast Benaderet as Cousin Pearl Bodine in the series and Benaderet appeared on the series' pilot and virtually every episode during the show's first season. Cousin Pearl and her daughter, Jethrine, moved into the Clampett mansion with the rest of the Clampett kin late in the first season, but Henning decided to cast Benaderet in the lead in his next series, Petticoat Junction, debuting in September 1963 (the female Bodines mysteriously disappearing from the Clampett estate for the show's second season.) Petticoat Junction proved an enormous hit and was a top ten program for several years. Oddly, Benaderet had done a radio variation of Green Acres with Gale Gordon beginning in 1950 called Granby's Green Acres, and the series later became a television spinoff of Petticoat Junction, with Eva Gabor playing the counterpart of Benaderet's part in the TV series and Benaderet herself showing up occasionally as her Petticoat Junction character (Eddie Albert took over Gale Gordon's role as the lawyer who moves to the country to become a farmer).

Benaderet also played a part in another iconic television series of the 1960s, cast as the voice of Betty Rubble on the cartoon series The Flintstones which debuted in 1960. Benaderet resigned from the animated series in 1964 due to her work load on her own program and Betty would be voiced by Gerry Johnson for the remainder of the series' run.

Benaderet became ill in 1967 which led to her leaving Petticoat Junction in what was hoped would be a temporary retirement. Rosemary DeCamp was brought in to play Aunt Helen in scripts obviously written for Benaderet's character Kate. Benaderet, however, was well enough only to make two additional appearances on the show. After her death, June Lockhart was brought in to play a female doctor who set up practice at the Shady Rest hotel and thus became the show's surrogate mother figure.

Bea Benaderet died from lung cancer at the age of 62, and her body was interred in the Valhalla Memorial Park Cemetery in North Hollywood, Los Angeles, California, next to her second husband, Eugene Twombley, who died of a heart attack only a week after the death of his wife. Mr. Twombley was a sound effects man for many radio and television shows, including The Jack Benny Program, on which Bea Benaderet was a regular.

Her son, Jack Bannon (whose father was Bea's first husband, Jim Bannon), later became a regular on Lou Grant.