Wickes was born in St. Louis, Missouri of German and Irish Protestant extraction in 1910. She began acting in films in the late 1930s, and was also a member of the Orson Welles troupe on his radio drama Mercury Theatre of the Air. One of her earliest significant film appearances was in The Man Who Came to Dinner (1942), reprising her stage role of Nurse Preen. A tall (5'10"), gangling woman with a distinctive voice, Wickes would ultimately prove herself adept as a comedienne, but she first attracted attention in the film Now, Voyager (1942), as the wise-cracking nurse who helped Bette Davis' character during her mother's illness. The same year she had a large part in the Bud Abbott and Lou Costello comedy-whodunnit, titled Who Done It?. She continued playing supporting roles in films during the next decade, usually playing wisecracking characters.

In the 1950s she played regular roles in the television sitcoms Make Room for Daddy and Dennis The Menace, as well as appearing as Emma the housekeeper in the holiday classic White Christmas (1954), starring Bing Crosby, Danny Kaye, Rosemary Clooney and Vera-Ellen. She served as the live-action reference model for Cruella De Vil in Walt Disney film One Hundred and One Dalmatians (1961). A lifelong friend of Lucille Ball, she played frequent guest roles in each of Ball's television series, I Love Lucy, Here's Lucy and The Lucy Show.

She was also a regular on the Sid and Marty Krofft children's television show Sigmund and the Sea Monsters. By the 1980s, her appearances in television series such as M*A*S*H, The Love Boat, Kolchak: The Night Stalker and Murder, She Wrote had made her a widely recognisable character actress.

She appeared in the 1990 film Postcards From the Edge cast as Shirley MacLaine's mother.

However, she achieved the biggest success of her career in Sister Act (1992). As Sister Mary Lazarus, Wickes' portrayal of a very gruff, strict but vulnerable elderly nun contributed to the film's popularity, and she reprised the role in the sequel Sister Act 2: Back in the Habit (1993).

She appeared in the 1994 film version of Little Women before she became ill. She was hospitalized the following year suffering from numerous ailments, including renal failure, massive gastrointestinal bleeding, severe hypotension, ischemic cardiomyopathy, anemia and breast cancer (stage of cancer unknown), which cumulatively resulted in her death.

A registered Republican who had never married, Wickes left a large estate and made a $2 million bequest, in memory of her parents, for the "Isabella and Frank Wickenhauser Memorial Library Fund for Television, Film and Theater Arts".

Her final film role, voicing the gargoyle Laverne in the animated feature The Hunchback of Notre Dame was released posthumously in 1996.

Although the nature of their relationship is disputed, she was the longtime companion of playwright Abby Conrad for many years.

In 2004 Mary Wickes was inducted into the St. Louis Walk of Fame.