Amazonia National Park is located in southwestern Pará State in central Amazonia. The 10,707 km2 park is one of the largest protected areas in Brazil and is covered with pristine forests, but the region is threatened by dam construction projects. An incomplete mammal biodiversity inventory was conducted in the area during the late 1970s. Here, we present results of sampling from 7,295 live-trap nights, 6,000 pitfall-trap nights, more than 1,200 km of walking transect censuses, and approximately 3,500 camera-trap days, all conducted between 2012 and 2014. These sampling efforts generated a list of 86 known species of nonvolant mammals, making the park the single most species-rich area for nonvolant mammals both in the Amazon Basin and in the Neotropics as a whole. Amazonia National Park is a megadiverse site, as is indicated by its mammalian richness, which includes 15 threatened mammal species and 5 to 12 new species of small mammals. As such, it merits being a high-conservation priority and should be an important focus of Brazilian authorities’ and the international scientific community’s conservation efforts. A comprehensive conservation plan is urgently needed, especially given the ecological threats posed by dam construction.