Abstract : Escherichia coli is a major etiological agent of Urinary tract infection (UTI) worldwide. People of all ages and gender are prone to UTI, but age over 20 years and women are more susceptible. The purposes of this study were to identify the E. coli isolates from suspected UTI samples and determine the prevalence and antimicrobial susceptibility pattern of the isolates. A total of 200 midstream urine samples were collected from suspected UTI patients between January 2016 and May 2016 from a tertiary care hospital. Presence of E. coli isolates in 138 samples was confirmed after microbiological and biochemical tests. Antimicrobial susceptibility testing of E. coli isolates revealed high level of resistance to Nalidixic Acid, Cefotaxime, and Ceftazidime. High level of sensitivity was found to Amikacin, Tobramycin, Netilmicin which can be used as effective antimicrobials, but before that clinician should determine antimicrobial susceptibility of UTI pathogen of suspected patients. To avoid antibiotic resistance, use of antibiotics should be monitored and drug regulations must be strictly followed