Buzzi was born in Westerly, Rhode Island, although she would later claim to have been born in Wequetequock, Connecticut—perhaps because it sounded funnier. She is the daughter of an Italian sculptor who specialized in making tombstones.

Ruth Buzzi was one out of only four people to appear in every episode of Laugh-In. Her signature character was the frowzy spinster "Gladys Ormphby," clad in brown with her bun hairdo covered by a visible hairnet. The character was a well-balanced and believable mix, defending her virginal purity and honor on the one hand, and desperately seeking wild and amorous romance on the other. The "defense" came from her lethal purse, with which she would flail away at anyone who sought to take advantage of her. On Laugh-In, Gladys most often appeared as the unwilling object of the advances of Arte Johnson's "dirty old man" character "Tyrone."

Buzzi, as "Gladys," later became a regular part of Dean Martin's "Celebrity Roasts," usually punishing Martin for his insults about her unappealing looks and romantic prospects. In one such exchange, Gladys accusingly questioned Martin about who had been chasing her around a hotel room in the wee hours; Martin's response, "The Exterminator," earned him a beating as he broke up laughing along with the audience. Gladys then declared to the audience that, when Martin and other men looked at her, only one thing came to their minds. Martin, still laughing, could barely get out the answer "Rabies!" which earned him an even fiercer beating from Gladys.

Ruth Buzzi also appeared on Sesame Street, You Can't Do That on Television (a Canadian production), and numerous other television shows. She was also a voice actress for The Smurfs. As of 2004 she was still appearing in movies.