Both cross-sectional study and field trial were undertaken from November 2018 to March 2019 with the objectives of estimating seasonal prevalence of bovine trypanosomosis, apparent density of Glossina and other biting flies and to assess the efficacy of diminazine aceturate and isometamidium chloride against most prevailing trypanosome spp in Assosa and Bambasi districts of Benishangul Gumuz region. One-stage cluster sampling strategy was used to select study animals; traps were deployed to collect Glossina species and an abbreviated 28-day field protocol based on treatment of naturally infected cattle was used to assess the efficacy of trypanocidal drugs. Entomological survey result revealed that only one species of Glossina (Glossina morsitans submorsitans) and three genera of other biting flies (Stomoxys, Tabanus and Haematopota) were captured and identified. The apparent density of Glossina was 0.31 fly per trap per day in the late rainy and 0.13 fly per trap per day in the dry seasons, with statistically significant difference (P<0.05). In the parasitological survey, blood samples were collected from ear vein of 1,562 head of cattle, 790 in the late rainy season and 772 in the dry period and examined with buffy coat technique; the overall prevalence of trypanosomosis was 7.7% in the late rainy season and 4.8% in the dry period with significant variation (P<0.05). The risk factors; district in the laterainy season as well as season were significantly associated (P<0.05) with bovine trypanosomosis while rural kebele, age, sex, body condition scores were not significant. Three species of trypanosomes were detected during the study, namely T. congolense (64%), T. vivax (21.3%), T. brucei (1.6%) and mixed infection (13.1%) in the late rainy season and T. congolense (75.7%), T. vivax (13.5%) and mixed infection (10.8%) in the dry season; T. congolense being the predominant species followed by T. vivax. The mean Paavked cell volume of trypanosome infected cattle (23.05±3.40) was significantly lower (P<0.05) compared to that of non-infected ones (27.73±4.61) in the late rainy season; similarly, it was significantly lower (P<0.05) in parasitemic cattle (23.54±2.58) than aparasitemic ones (28.15±4.58) in the dry season. Treatment failure was detected in 19/30(63.3%) of cattle treated with 3.5mg/kg b.w diminazene aceturate 14 days post treatment. Re-treatment of T. congolense positive cattle that had failed diminazene aceturate treatment with 7mg/kg b.w had still resulted in a treatment failure of 4/17(35.3%). Similarly treatment failure was observed in 8/30(26.7%) and 8/22(36.4%) of cattle treated with isometamidium chloride at dose of 0.5mg/kg b.w 14 and 28 days post treatment resulting in cumulative treatment failure of 16/30(53.3%). Trypanosoma congolense accounted foremost treatment failure for the two drugs. Assosa district (Megele 38) had higher treatment failure when compared to Bambasi district (Nebar keshimando) for the two typanocidal drugs; however, no significant variation was observed (P>0.05). The entomological survey and the parasitological findings revealed that bovine trypanososmosis is widely distributed and endemic in Assosa and Bambasi districts of Benishangul Gumuz region and also fauilre of trypanocidal drug efficacy was found to be a constraining factor to control the disease using curatve and prophylactic drugs; hence designing participatory and integrated control measures including regular surveillance, community based vector prevention and control measures should be implemented to mitigate the problem. Keywords: Benishangul Gumuz, Bovine, Epidemiology, Glossina morsitans submorsitans, Season, Trypanosomosis,