Born in Toowong, Australia, Dart was educated at the universities of Queensland and Sydney where he qualified as a physician in 1917. After a short period (1919–22) at University College, London, Dart moved to South Africa to serve as professor of anatomy at the University of the Witwatersrand, Johannesburg, a post he held until his retirement in 1958.

In 1924 Dart was privileged to make one of the great paleontological discoveries of the century, the Taung skull. For this he was indebted to his student Josephine Salmons who brought him in the summer of 1924 a fossil collected from a mine at Taung, Bechuanaland. Dart named it Australopithecus africanus, meaning southern African ape, and declared it to be intermediate between anthropoids and man. Such a claim was far from acceptable to many scholars at the time who, like Arthur Keith, dismissed the skull as that of a young anthropoid. Other and older australopithecine remains were later discovered by Robert Broom in South Africa, East Africa, and Asia, making it clear that they were in fact hominid. It is still however a matter of controversy whether Australopithecus lies in the direct line of descent to Homo sapiens or whether it represents a quite separate and unsuccessful evolutionary sideline.