Mammals represent the largest-bodied elements of the world’s surviving megafauna and provide several key ecosystems services, yet their populations are often under steep decline throughout the tropics. Anthropogenic defaunation is one the most important contemporary threats to modern mammal faunas. Although the Atlantic Forest biome of South America shows several clear signs of defaunation, the extent to which this biome has lost its mammal fauna remains poorly understood. Here, we collate and analyze a comprehensive body of secondary data to quantitatively assess the spatial patterns of defaunation of all medium- to large-bodied Atlantic Forest mammals which were then classed by morpho-ecological traits. We used a Defaunation Index, which was scaled-up to the entire biome using kriging interpolation, to examine the integrity of site-specific mammal faunas. We further use environmental and socioeconomic predictors to explain the drivers of defaunation. Our results show high levels of defaunation (>0.5) for most of the Atlantic Forest. Apex predators, other carnivores, large-bodied mammals and large herbivores were among the most defaunated functional groups. Remaining native vegetation cover, forest fragment size, and the largest neighboring forest remnant were the main negative predictors of defaunation. We conclude that medium- to large-bodied Atlantic Forest mammals are under high levels of threat due to historical population losses that continue today. A conservation action plan thus becomes imperative to prevent this biome from becoming an even “emptier forest”, severely compromising patterns of diversity, ecological processes and ecosystem functioning.