Introduction Antibiotic resistance is increasing in gram-negative bacteria that cause urinary tract infections. The aim of this study was to evaluate antibiotic resistance pattern and frequency of broad-spectrum β-lactamases (ESBLs) among gramnegative bacteria isolated from urine samples referred to clinical laboratories in Bandar Torkaman, Iran. Materials and Methods In this study, 138 positive urine samples were collected from patients with suspected urinary tract infection. In positive samples, gram-negative europathogens were identified using standard microbiological and biochemical tests and their antibiotic resistance patterns were determined by propagation in the disk method. Then, a phenotypic confirmatory test was performed for the detection of ESBLs producers. Results 96 samples were positive for urinary infection due to gram-negative bacteria. The most isolated cases were: Escherichia coli (88. 5%), Klebsiella pneumonia (5. 2%), Enterobacter cloace (4. 2%) and pseudomonas aerouginosa (2. 1%). The E. coli isolates showed the highest resistance to ampicillin, nitrofurantoin and cefalotin and the lowest resistance to norfloxacin. Of the 35 isolates resistant to cefotaxime, 29 isolates were positive for ESBLs. Conclusion In this study, the most common cause of UTI was E. coli (88. 5%). The frequency of ESBLs in E. coli isolates was similar to the results of other studies but it was very high inK. pneumonia isolates. The results of this study indicate an increasing trend of resistance to Extended-Spectrum cephalosporins. Therefore, antibiogram test before the antibiotic prescription could be a retional strategy to avoid further antibiotic resistance.