AbstractAmong the bacterial infections that impair the health status of marine mammals, those caused by Brucella spp. are the most reported worldwide. Brucella infections in marine mammals can result in acute or chronic disease and are associated with variable clinical outcomes, depending on the organ involved during the infectious process, infection route, host immunity, and strain pathogenicity. Asymptomatic infections may also occur. The current study expands the investigation of Brucella infection in northeast Brazil by analyzing 19 dead, stranded cetaceans and 52 Antillean manatees Trichechus manatus manatus. The manatees included 8 dead, captive manatees and 44 live specimens, of which 10 were analyzed only after reintroduction into the wild as part of a rehabilitation program, 9 were analyzed both while in captivity or semi‐captivity and after reintroduction, 20 were sampled only in captivity or semi‐captivity, and 5 were free‐living manatees. Serological tests were used to screen for antibodies against smooth Brucella spp. Whole blood, swabs, and tissue samples were screened for Brucella spp. DNA by PCR. Samples with positive PCR results were cultured for Brucella spp. isolation. All manatees yielded negative results in serological and molecular tests. Brucella spp. DNA was detected in the kidney of one adult Guiana dolphin Sotalia guianensis exhibiting necrosis in the liver. No growth of Brucella spp. was observed via microbiological culturing. This study is the first report of Brucella spp. DNA detection in cetaceans in the state of Pernambuco, and it highlights the importance of conducting systematic monitoring for the presence of Brucella infection in marine mammals along the Brazilian coast, especially in the northeast region, where several cases have been reported.