The extinction of late Quaternary megafauna in South America has been extensively debated in past decades. The majority of the hypotheses explaining this phenomenon argue that the extinction was the result of human activities, environmental changes, or even synergism between the two. Although still limited, a good chronological framework is imperative to discuss the plausibility of the available hypotheses. Here we present six new direct AMS 14C radiocarbon dates from the state of São Paulo (Brazil) to further characterize the chronological distribution of extinct fauna in this part of South America. The new dates make evident that ground sloths, toxodonts, and saber-toothed cats lived in the region around the Pleistocene/Holocene transition, and in agreement with previous studies, also suggest an early Holocene survival for the ground sloth Catonyx cuvieri. Taken together with local paleoclimatic and archaeological data, the new dates do not support hunting or indirect human activities as a major cause for megafauna extinction. Although more data are required, parsimony suggests that climatic changes played a major role in this extinction event.