The continued emergence of ESBLs presents diagnostic challenges to the clinical microbiol-ogy laboratories, which should be aware of the need for their detection by accurately identi-fying the enzymes in clinical isolates. The importance of the infections due to Extended-Spectrum β-Lactamase-producing Escherichia coli and Klebsiella species (ESBL-EK) has been increasingly recognized in recent years. ESBL-EK infections are of clinical concern, because few antimicrobials are available as therapeutic options. Efforts to maintain current therapeutic option for such infections are essential. Total 200 screened isolation of E. coli and Klebsiella species obtained from June 2006 to 31st December 2007 from 3146 various clinical samples such as pus; urine, blood, CSF, ear discharge, pleural fluid & sputum were included in the study. All isolates were identified and confirmed by standard conventional methods. Isolates with resistance or decreased susceptibility to third generation cepha-losporins (as per the criteria of NCCLS guidelines by Kirby-Bauer methods) were selected as ESBL producers. ESBL production where detected by Double disk synergy test (DDST) and Inhibitory Potentiated disk diffusion test (IPDDT). Of the 200 isolates, 117(58.5 %), 83(41.5%) were identified as E.coli and Klebsiella respectively and showed maximum resis-tance against Cefpodoxime (100%). By DDST test 92.6% & 89.1%, 66.7% & 71.7%, 88.8% & 71.7% showed synergy between Ceftazidime, Cefotaxime, Ceftriaxone with Clavulanic acid by Klebsiella species and E.coli respectively. The incidence of ESBLs among E. coli and Klebsiella species were 44.4% (52) & 36.1% (30) respectively by IPDDT test and detect an additional 4.5%(9) ESBL isolates than DDST test. Medicine 56.1% & 44.8% and Sur-gery 21% & 31% wards showed maximum percentage of ESBLs producers E. coli and Klebsiella respectively. ESBL-producing E. coli and Klebsiella species infections have a sig-nificant impact on several important clinical outcomes and efforts to control outbreaks of such infections should emphasize judicious use of all antibiotics as well as barrier precau-tions to reduce spread.