Brucellosis is a zoonosis. The aim of this study was to check the health status of felids kept at the zoo of the Federal University of Mato Grosso, Cuiabá, Brazil. Blood samples were taken from seven Puma Puma concolor, five Jaguar Panthera onca, four Ocelot Leopardus pardalis and one Lion Panthera leo. Brucellosis serology and polymerase chain reaction were performed. The presence of antibodies and DNA of Brucella abortus and Brucella canis were not detected in P. leo. The amplification of DNA from B. abortus and B. canis was observed in the four Ocelot. DNA from B. abortus was detected in one Jaguar, and DNA from B. canis in two Puma and one Jaguar. Serology for B. abortus was positive in two Puma and two Jaguar, while B. canis was observed in one Ocelot and two Jaguar. Although not considered a source of infection to staff, multidisciplinary studies should be carried out to elucidate the role of domestic animals in the epidemiology of this zoonosis.