Brucellosis is a zoonotic disease caused by Brucella species through infected domestic animals. In endemic areas, certain occupations such as veterinarians, butchers, and slaughterhouse workers are considered high risk for brucellosis. We evaluated the seroprevalence of brucellosis in people with high-risk occupations in three cattle slaughterhouses in Peru. A questionnaire including demographic data, length of employment, and the use of personal protective equipment was completed for each of them, then blood samples were obtained and analyzed using the ELISA test to detect anti-Brucella IgM antibodies. Overall, 49 of 173 (28, 32%) participants exhibited seroreactivity for brucellosis. Headache, joint pain, night sweats, fever, and anorexia were common symptoms in seropositive cases. Furthermore, 63.27% of seropositive participants were asymptomatic. The prevalence of brucellosis in these occupational groups in relation to the absence of clinical manifestations in a significant number of them was high, thus periodic clinical examinations in these groups appear to be essential to the brucellosis surveillance system. Regular and consistent use of equipment should be encouraged of personal protection, especially goggles, among cattle slaughterhouse workers for reduce brucellosis infection.