BackgroundUrinary tract infection (UTI) remains to be one of the most common infectious diseases diagnosed in developing countries. And a widespread use of antibiotics against uropathogens has led to the emergence of antibiotic resistant species. A laboratory based cross-sectional survey was conducted in Shashemene referral hospital to determine the prevalence and antibiotic susceptibility of uropathogens.MethodsWe have collected 384 clean catch mid-stream urine samples from all suspected UTI outpatients using sterile screw capped container. The urine samples were cultured and processed for subsequent uropathogens isolation. The isolated pure cultures were grown on BiOLOG Universal Growth agar (BUG) and identified using GEN III OmniLog® Plus ID System identification protocols. The identified species were then exposed to selected antibiotics to test for their susceptibility.ResultsThe overall prevalence of urinary tract infection in the area was 90.1%. Most frequently isolated uropathogen in our study was Escherichia coli (39.3%). While, Staphylococcus species (20.2%), Leuconostoc species (11.4%), Raoultella terrigena/Klebsiella spp./ (8.4%), Salmonella typhimurium (6.3%), Dermacoccus nishinomiyaensis (6.3%), Citerobacter freundii (5.2%) and Issatchenkia orientalis/Candida krusei/ (2.7%) were the other isolates. We find that the relationship between uropathogens and some of UTI risk factors was statistically significant (P < 0.05). Gentamicin was the most effective drug against most of the isolates followed by chloramphenicol and nitrofurantoin. In contrast, amoxicillin, vancomycin and cephalexin were the antibiotics to which most of the isolates developed resistance.ConclusionUrinary tract infection was highly prevalent in the study area and all uropathogens isolated developed a resistance against mostly used antibiotics.Electronic supplementary materialThe online version of this article (10.1186/s12879-017-2911-x) contains supplementary material, which is available to authorized users.