Urinary tract infection (UTI) is the most common infection in both community and hospital patients. In majority of the cases, empirical antimicrobial treatment is practiced before the laboratory results of urine culture. Thus, antibiotic resistance may increase in urinary bacterial pathogens due to improper use of drugs. This study was designed to find out the etiological agents of UTI and their prevalence, and to determine the antimicrobial susceptibility pattern of the bacterial pathogens isolated from urine culture. This study was conducted in Kathmandu Model Hospital, Kathmandu, Nepal from April to October, 2009. Midstream Urine samples from 1323 patients suspected of UTI were analyzed by microscopy, and conventional semi-quantitative culture technique for the significant growth. Antimicrobial susceptibility test was performed for the isolates by Modified Kirby-Bauer disk diffusion method. Data were analyzed using SPSS software window version 16. The overall prevalence of UTI was found to be 18.89%. The most frequent causative organisms isolated were Escherichia coli (82.30%), Enterococcus faecalis (5.60%), Citrobacter freundii (3.60%), Enterobacter aerogenes (2.40%), Coagulase Negative Staphylococci (2.40%), Pseudomonas aeruginosa (1.20%), Proteus mirabilis (0.8%), Klebsiella pneumoniae (0.4%), and Staphylococcus aureus (0.4%). Nitrofurantoin and Amoxycillin were found to be the most effective antibiotic against gram negative and gram positive isolates respectively. E. coli was found to be the most common etiological agent of UTI and Nitrofurantoin was the most effective drug among the isolates.