She was raised in the Baptist faith of her mother. While the children were still young, the Gardners lost their property, forcing Jonas Gardner to work at a sawmill and Molly to begin working as a cook and housekeeper at a dormitory for teachers at the nearby Brogden School. When Gardner was seven years old, the family decided to try their luck in a larger city, Newport News, Virginia, where Mollie Gardner found work managing a boarding house for the city's many shipworkers. While in Newport News, Gardner's father became ill and died from bronchitis in 1938, when Ava was 15 years old. After Jonas Gardner's death, the family moved to Rock Ridge near Wilson, North Carolina, where Mollie Gardner ran another boarding house for teachers. Gardner attended high school in Rock Ridge and she graduated from there in 1939. She then attended secretarial classes at Atlantic Christian College in Wilson for about a year.
Gardner was visiting her sister Beatrice in New York in 1941 when Beatrice's husband Larry Tarr, a professional photographer, offered to take her portrait. He was so pleased with the results that he displayed the finished product in the front window of his Tarr Photography Studio on Fifth Avenue.
After five years of bit parts, mostly at MGM and many of them uncredited, Gardner came to prominence in the Mark Hellinger-produced smash-hit film noir The Killers (1946), playing the femme fatale Kitty Collins.
Other films include The Hucksters (1947), Show Boat (1951), The Snows of Kilimanjaro (1952), Lone Star (1952), Mogambo (1953), The Barefoot Contessa (1954), Bhowani Junction (1956), The Sun Also Rises (1957), and On the Beach (1959). A particularly notable role was in The Barefoot Contessa as the doomed beauty Maria Vargas, a fiercely independent woman who goes from Spanish dancer to international film star with the help of a Hollywood director played by Humphrey Bogart, with tragic consequences.
John Huston chose Gardner for the part of Sarah, the wife of Abraham (played by George C. Scott), in the Dino De Laurentiis production The Bible: In the Beginning..., which was released in 1966. In a 1964 interview, she explained why she accepted the role:
He [Huston] had more faith in me than I did myself. Now I'm glad I listened, for it is a challenging role and a very demanding one. I start out as a young wife and age through various periods, forcing me to adjust psychologically to each age. It is a complete departure for me and most intriguing. In this role, I must create a character, not just play one.