Studies on human brucellosis in Rwanda are scarce, and the incidence is likely to be higher than official estimates. In a recent study using Rose Bengal Test (RBT) on women who had aborted or had still births in Huye district, 25% were Brucella seroprevalent. Thus, purpose of the present study was to investigate the Brucella seroprevalence in patients presenting with the key signs and symptoms of brucellosis. Cross-sectional study was done in Nyagatare District in the Eastern Province of Rwanda. A total of 198 patients were recruited from Nyagatare District Hospital, blood samples were collected, and sera analyzed with RBT. A questionnaire was used to explore the risk factors. A total of 12 patients (6.1%; 95% confidence interval [CI] = 0.662-7.820) were Brucella seropositive. Infection was significantly associated with drinking unboiled milk (odds ratio [OR] = 8.3; 95% CI = 2.4-29.2) and having had recurrent fever (OR = 5.6; 95% CI = 1.5-21.3). Drinking unboiled milk is a risk factor for Brucella infection. Provision of adequate resources and trainings to staff in brucellosis diagnosis is needed to reduce recurrence of fevers probably because of misdiagnosis. Public awareness creation on transmission routes of brucellosis is to be intensified.