Background and Objectives: This study was designed as a retrospective study on urine samples during three years in Shaheed Mostafa Khomeini Hospital to determine demographic characteristics of patients with urinary tract infection (UTI), microbial etiology, and susceptibility of isolated bacteria to antibiotics. Materials and Methods: All urines fulfilling the criteria for significant bacteriuria (>104 colonyforming units/ml of urine) were included in the study. Isolation and identification of bacteria was performed by standard method and susceptibility testing was determined by disk diffusion method according to NCCLS guideline. A total of 909 patients with urinary tract infection were enrolled in this study. Results: Mean age of the patients was 53.2 years. In addition, females were affected more often than males (female/male sex ratio was 2.22). Meanwhile, considering all strains, 79.5% were Gram-negative bacilli and 67.7% were Enterobacteriaceae. Furthermore, E.coli and Klebsiella spp represented the most common Gram-negative and Enterococci and S. aureus represented the most frequent Gram-positive isolates. The four most frequently isolated bacteria were E. coli (52.1%), Enterococci (10.5%), klebsiella spp. (10.3%), and pseudomonas spp. (9.4%). In addition, E. coli was significantly more common in females (56.6%) than in males (42.2%) and in outpatients (57.4%) than in inpatients (47.4%). The proportion of pseudomonas spp. was significantly higher in males (17.7%) than in females (5.6%). Enterococci were significantly more common in inpatients (12.5%) than in outpatients (8.4%). Altogether, the rate of susceptibility of all UTI pathogens was very low to ampicillin (6.9%) and high to cefotaxime (83.6%) and ciprofloxacin (78.2%). Urinary pathogens isolated from female patients and outpatients were more susceptible to most of examined antibiotics than those isolated from males and inpatients. Conclusion: It was found out that degrees for antibiotic resistance of urinary pathogens are alarming and show the necessity of keeping up the monitoring of antibiotics susceptibility in UTI isolates and restricting antibiotic consumption in our population.