She married Stephen Vanderburgh Harkness, a harnessmaker of Cleveland, in 1851. They were parents of Charles William Harkness and Edward Stephen Harkness. Harkness senior invested with John D. Rockefeller and became the second-largest shareholder in Standard Oil.
In 1918 Anna Harkness established the Commonwealth Fund with an initial gift of $10 million, and made her son Edward its president. From this fund the Harkness Fellowships were established, and St. Salvator's Hall at the University of St Andrews, the Butler Library at Columbia University and many of the undergraduate dorms at Harvard University and Yale University were built.
Anna Harkness donated another $3 million to Yale in 1920 to increase faculty salaries.
Anna Harkness also made gifts to Hampton and Tuskegee Institutes, the New York Public Library, the Museum of Natural History in New York, the Metropolitan Museum of Art, the New York Zoological Society and Columbia University College of Physicians and Surgeons.