Brucellosis is a zoonotic disease primarily affecting animals and accidentally exposed humans. In Eritrea, brucellosis endemicity is proven by several sero-prevalence studies in domestic animals and occupationally exposed humans. However, there is a gap of information on the commonly occurring brucella species and cause of human/animal brucellosis. The objective of this study is to identify brucella species in sheep and goats which possibly pose risk to human brucellosis. Out of a total 71 serum and 71 vaginal swab samples collected from sheep and goats, eight serum samples by Rose Bengal plate test (RBPT) and 4 vaginal swabs by polymerase chain reaction (PCR) were found positive for brucellosis. Combinatorial PCR detected Br. melitensis from the four PCR positive samples. Questionnaire interviews collected from 68 confirmed brucellosis patients showed that habits of drinking unpasteurized yoghurt and raw milk and handling of aborted materials were common. This investigation study suggests, Br. melitensis could be the major cause of brucellosis in humans and animals in Maekel region Eritrea. Therefore, a large-scale epidemiological study is recommended to confirm the true extent of the problem in animals and humans.