To evaluate the prevalence and possible risk factors for brucellosis caused by Brucella abortus in water buffaloes in the state of Pará, Brazil, 3,917 female buffalo serum samples from pregnant and non-pregnant animals were examined: 2,809 from Marajó Island and 1,108 from the mainland. The buffered acidified plate antigen (BAPA) screening test positively diagnosed 4.8% (188/3,917) of the animals with brucellosis, and the 2-mercaptoethanol (2-ME) confirmatory test affirmed 95.7% (180/188) of the results. The brucellosis prevalence was 4.17 times greater in mainland animals than on Marajó Island, with the highest prevalence in Tailândia (11.30%) and Paragominas (12.38%). Brucellosis seroprevalence was significantly influenced (p < 0.05) by reproductive status, with pregnant females being most vulnerable. These results demonstrate that brucellosis infection is active in the Brazilian region containing the largest buffalo population and that this disease poses a threat to public health and buffalo production in Pará.