Bovine brucellosis is endemic in Rwanda; however, little information is available on seroprevalence and risk factors. Therefore, a cross-sectional study was conducted among cattle farmed at the wildlife-livestock-human interface (n = 1691) in five districts and one peri-urban district (n = 216). Cattle were screened using the Rose Bengal test, then the results were confirmed by indirect enzyme-linked immunesorbent assay. Potential risk factors were determined with a questionnaire and analyzed for their association with seropositivity. In all districts, the animal and herd-level seroprevalence was 7.4% (141/1907) and 28.9% (61/212), respectively, 8.3% (141/1691) and 30.9% (61/198) at the interface, and 0.0% (0/216) in peri-urban areas. Among the potential risk factors, old age (≥5 years), cattle farmed close to wildlife, herds of cattle and small ruminants, history of abortions, and replacement animals were significantly associated with brucellosis (p < 0.05). Low awareness of zoonotic brucellosis, assisting calving without biosafety protection, drinking raw milk, and manual milking were each observed in more than 21.7% of cattle keepers whose herds were seropositive. This study confirmed brucellosis endemicity in cattle farmed close to wildlife in Rwanda, suggesting the need to focus control efforts in these areas. Educated farmers with a high awareness of zoonotic brucellosis had low bovine brucellosis seropositivity, which emphasizes the importance of education.