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Aquatic mammals can act as sentinels of emerging and resurging pathogens in the environment. Brucella spp. and Leptospira spp. are 2 zoonotic pathogens relevant to aquatic mammals, and their detection can be used to assess pathogen exposure. In this study, serum from 84 individuals-63 cetaceans (families Iniidae, n = 37; Delphinidae, n = 22; and Kogiidae, n = 4) and 21 West Indian manatees Trichechus manatus-was tested by the Rose Bengal Test (RBT) and a commercial competitive enzyme-linked immunosorbent assay (c-ELISA) for detecting Brucella spp. antibodies, and the microscopic agglutination test (MAT) for screening Leptospira spp. exposure. Overall, 4.8% (3/63) of cetaceans were positive by RBT and 15.9% (10/63) by c-ELISA for Brucella spp. Serum from 8 c-ELISA positive cetaceans (with available serum) was further tested via serum agglutination test (SAT) and 1 individual was positive. c-ELISA was more sensitive than RBT. Exposure to Brucella spp. was found in 5 cetacean species: Clymene dolphin Stenella clymene, short-finned pilot whale Globicephala macrorhynchus, pygmy killer whale Feresa attenuata, melon-headed whale Peponocephala electra and Atlantic bottlenose dolphin Tursiops truncatus in the Atlantic Ocean, Brazil, expanding the range of known Brucella seropositive aquatic hosts. No evidence of Brucella spp. exposure was found in Iniidae and Kogiidae odontocetes and manatees. Antibodies against Leptospira spp. were not detected in cetaceans and sirenians by MAT. These results contribute to the evaluation of different Brucella spp. serological methods in cetaceans and manatees and highlight the epidemiology of zoonotic pathogens in aquatic mammals of the southwestern Atlantic Ocean and the Amazon basin.