Some of gram negative Bacilli such Klebsiella pneumoniae and Escherichia coli are a relevant opportunistic pathogen that accounts for nosocomial infections. Urinary Tract Infection (UTI) is a on of the most prevalent infection in the worldwide and. Antibiotic resistance in Bacteria is one of the emerging health related problem in the world nowadays. The search were laboratory and performed in Azzahra and Shariaty hospitals in 2009 year in Isfahan, according to statistical formula randomly selected 91 samples from urinary infections. Bacterial identification was performed with microbiological methods. ESBLs determine were in two section, screening and confirming testing were respectively included by Kirby-Bauer’s disc diffusion and Combining disc methods and survey antibiogram was performed with by Kirby-Bauer method. From 91 samples frequency of E.coli and K.pneumoniae strains was respectively 84/6% and 15/4%. From 77 isolated E.coli strains: 76.6% from out hospitalized and 23.4% from hospitalized and from 14 isolated K.pneumoniae strains: 57.1% from out hospitalized and 42.9% from hospitalized patients. According to antibiogram result respectively 59.2%, 54.9%, 30.3%, 27.8%, 19.5% and 16.7% of E.coli strains into Co-Trimoxazole, Nalidixic acid, Ciprofloxacin, Gentamicin, Ceftazidime, Nitrofurantoin, and respectively 75%, 50%, 40%, 44.5%, 37.5%, 37.5%, 22.3% and 0% of K.pneumoniae strains were resistant into Ampicillin, Co-Trimoxazole, Nitrofurantoin, Ceftazidime, Amikacin, Cephotaxime, Imipenem and Ciprofloxacin. The result of this study showed that the rate of nosocomial UTI in hospitalized patients. According to increase of resistance to the common usage drugs in UTI gram-negative bacteria, correct usage of antibiotics is recommended.