β-Lactam antibiotics are commonly used to treat bacterial infections. Increased use of antibiotics, particularly the third generation of cephalosporins, has been associated with the emergence of ESBL producing bacteria, most commonly in E. coli. And Klebsiella pneumonia. Infection due to ESBL producers range from uncomplicated urinary tract infection to life threatening sepsis. This study was undertaken to assess the prevalence of ESBL producing E.coli and Klebsiella isolates. Patients taking treatment in P.D.U. Hospital and Medical College, Rajkot- a tertiary care hospital in Gujarat, India, were included in study. During the half year period from July 2014 to December 2014, a total of 600 E. coli and Klebsiella isolates from various clinical specimens were included in the study. The isolated organisms of E. coli and Klebsiella were characterized for their production of ESBL. Out of 600 E. coli and Klebsiella isolate, E. coli were 224 (37.3% and Klebsiella spp. 376 (62.7%). The overall rate of production of ESBL (by phenotypic confirmatory test with double disc diffusion method) in total isolates of E. coli and Klebsiella spp. is 22.5% (135/600). The rate of production of ESBL in E. coli is 29.9% (67/224) and in Klebsiella spp. is 18.1% (68/376). ESBL producing E. coli and Klebsiella showed high prevalence in present study. Routine laboratory testing for ESBL is needed in order to optimize antibiotic management to reduce ESBL associated morbidity & mortality.