Born Mae Kwestel in New York City, Questel won a talent contest at the age of 17, and began performing in vaudeville. She was seen by animator Max Fleischer, who was looking for an actress to provide the voice for his Betty Boop character. Questel's "Boop-boop-a-doop" routine, done in a style similar to that of the song's originator, Helen Kane, while at the same time evoking something of the naughty allure of film star Clara Bow, was exactly what Fleischer had been looking for. From 1930 until 1939 Questel provided the voice of Betty Boop in more than 150 animated shorts. During the 1930s she released a recording of "On The Good Ship Lollypop" which sold more than 2 million copies.

From the mid 1930s Questel also provided the voice for Olive Oyl in the Popeye animated shorts. She based Olive's nasal vocal style on that of the legendary actress ZaSu Pitts, and ultimately played the role for more than twenty years.

Questel made her first on-screen appearance in the 1960s and was widely seen as one of Fanny Brice's mother's card-playing friends at the start of Funny Girl (1968). She appeared in Zelig (1983) and memorably (as a celestial apparition) in New York Stories (1989).

Her final film appearance was in National Lampoon's Christmas Vacation (1989). She also provided the voice for her old character Betty Boop in a cameo appearance in Who Framed Roger Rabbit (1988). She achieved perhaps her greatest visibility in television commercials for various household products, most notably as "Aunt Bluebell", pitching Scott Towels.