In a study of three high-risk occupational groups using Rose Bengal and complement fixation tests, the highest prevalence (7.1%) was found among dairy farm workers and owners in randomly selected dairy-cattle farms, followed by veterinary personnel (4.5%) and inhabitants in pastoralist areas (3.0%). There was no evidence for significant differences between the three populations. Among dairy farm workers, a higher risk was associated with the presence of sheep in the farm (OR = 13.2, CI = 2.2–76.7). In the pastoral area, a high risk was linked to having close contact with animals (OR = 6.32, CI = 0.88–∞), while a reduced risk was seen for contact with cattle (OR = 0.18, CI = 0–1.30). Symptoms suggestive of brucellosis were more commonly observed among the dairy farm workers, mainly found in the highlands, than among the pastoralist area inhabitants, where malaria is prevalent. The study documents not only the presence of serological and clinical evidence of human brucellosis, but also risk factors related to it in Eritrea, for the first time.