Background: ASB is a common problem in pregnant women and about 40% of women with untreated asymptomatic bacteriuria during pregnancy develop pyelonephritis, which might lead to low birth weight, preterm, premature rupture of membranes and preterm labour. Therefore, this study aimed to assess the prevalence of asymptomatic bacteriuria, antimicrobial susceptibility pattern of the isolates among pregnant women attending antenatal care clinic of Assosa General Hospital, western Ethiopia.Methods: A facility-based cross sectional study was conducted from January to February 2019. Well-mixed and uncentrifuged urine specimens obtained from the pregnant women was directly inoculated on cystine lactose electrolyte deficient agar (CLED) (Oxoid, Ltd, England) media by streak plate method. Bacterial isolates were identified as per the standard bacteriological procedure using colony characteristics, gram-staining, and series of biochemical tests. Antimicrobial susceptibility test was carried out by Kirby- Bauer disk diffusion technique on Muller-Hinton agar medium and the diameter of zone of inhibition was interpreted according toClinical Laboratory Standard Institute (CLSI) guidelinesResult: The overall prevalence of ASB among pregnant women in this study was 14.85%.E. coli was the most predominant isolate (50 %,) followed by K. pneumoniae (16.7%,), S. aureus (14.3%), coagulase negative staphylococci (CONS) (11.9%), and group B streptococci (GBS) (7.1%).Gram-negative bacteria were highly resistant for tetracycline (96.4%), ampicillin .Gram-positive bacteria were 100% sensitive for ceftazidime. Conclusion: Significant bacteriuria was observed in asymptomatic pregnant women. Therefore, routine laboratory diagnosis of ASB in pregnant women and providing appropriate treatment should be needed to reduce its complications.