Large and medium-sized mammals are essential to forest ecosystems as they are responsible for prey population control, seed dispersal and predation. A mammal survey was conducted in the Humaitá Forest Reserve (HFR), a 20 km² forest fragment located in the southeastern part of the state. The survey was carried out using census transects, camera traps, and occasional records such as bones and footprints. In all, 27 species of large to medium-sized mammals were recorded for the RFH, representing 48% of those expected for the region. Dasyprocta fuliginosa, Pecari tajacu and Didelphis marsupialis were the species with the highest number of records with camera traps. The occurrence of two species of cats (Leopardus pardalis and Puma concolor) and three endangered species (Callimico goeldii, Myrmecophaga tridactyla and Priodontes maximus) are important records for the RFH. The results may aid future research on the ecology, biogeography and conservation of mammals in the region.