Background: Malaria is the leading health problem in Ethiopia. The country has been prevented malaria vectors mostly using long-lasting insecticide-treated nets, the application of indoor residual spraying chemicals, and source reductions. Before interventions, identifying the responsible malaria vector in disease transmission (sporozoite rate) is very vital; hence, the present study was designed to assess species diversity and entomological inoculation rate of Anopheles mosquito in Bure district, Northwest Ethiopia. Methods: Adult mosquitoes were collected from July 2015 to June 2016 using the center for disease control and prevention light traps, pyrethrum spray catches, and artificial pit shelters. Mosquitoes were morphologically identified. Following this, An. gambiae s.l was identified molecularly. Head-thorax sporozoite infectivity of the adult female Anopheles mosquitoes was assessed using enzyme-linked immunosorbent assays. Results: Morphologically, nine species of the genus Anopheles were identified in the three villages, composed of Anopheles demeilloni, An. arabiensis, An. funestus, An. coustani, An. squamosus, An. cinereus, An. pharoensis, An. rupicolus, and An. natalensis. Of these species, An. demeilloni was the most predominant, whereas An. cinereus, An. rupicolus and An. natalensis were the least representative species (p < 0.0001). Greater number of adult Anopheles mosquitoes were collected in Shnebekuma, non-irrigated villages than non- irrigated village (Workmidr) and irrigated village (Bukta) (p < 0.0001). The overall Plasmodium infective rate (P. falciparum and P. vivax) in the district was 0.31%. The overall annual sporozoite rate in non-irrigated villages (Shnebekuma and Workmidr) was 0.35%, whereas zero in irrigated village (Bukta). The overall estimated EIR of Anopheles mosquitoes was 5.7 infectious bites /person /year for both P. falciparum and P. vivax in the district. The annual EIR Anopheles species in non-irrigated villages was 5.65 ib/p/y, which was higher than irrigated village (0 ib/p/y). Conclusions: Both the primary (An. arabiensis) and secondary (An. funestus and An. pharoensis) malaria vectors of Ethiopia were identified in the three villages. Three of Anopheles species, An. arabiensis, An. funestus, and An. coustani were found to be infected only in irrigated villages. Source reduction and proper usage of long-lasting insecticide nets and indoor residual spraying could be implemented in the non- irrigated villages to cut the vector abundance and EIR.