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Background Asymptomatic bacteriuria 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, 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 the antenatal care of Assosa general hospital, western Ethiopia. Methods A facility-based cross-sectional study was conducted from January to February 2019. Two hundred and eighty-three pregnant women with no symptoms of urinary tract infections participated in the study. 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 to Clinical Laboratory Standard Institute guidelines. Results The overall prevalence of asymptomatic bacteriuria among pregnant women in this study was 13.78% (i.e. 39 out of 283 urine samples were positive for bacterial isolates). E. coli was the most predominant isolate (53.8%) followed by K. pneumoniae (17.95%), S. aureus (15.4%), and coagulase-negative staphylococci (12.8%). Gram-negative bacteria were highly resistant to tetracycline (96.4%), and ampicillin (90.5%). Conclusion Significant bacteriuria was observed in asymptomatic pregnant women. A large number of the bacterial isolates were resistant to the commonly used antimicrobial drugs.