Background: Extended spectrum beta lactamase (ESBLs) are betalactamase capable of conferring bacterial resistance to the first, second and third-generation Cephalosporin's but these are inhibited by lactamase inhibitors such as clavulanic acid. The ESBLs are typically plasmidmediated enzymes, mostly produced by E. coli and Klebsiella pneumonia Enterobacteriaceae family. ESBLs enzymes were discovered in Germany in 1983 from Klebsiella pneumonia. ESBL-producing gram negative bacteria have been responsible for numerous outbreaks of infection throughout the several counties and region challenging infection control issues. Different phenotypic test for ESBL detection have been developed, which are easy to use and cost effective. Materials and Methods: In this study, 10 Urine samples were collected from Government Hospital, Dharmapuri, Tamil Nadu. Collected clinical samples were isolated and identified by standard microbiological method. The antibiotic susceptibility testing was performed by the disc-diffusion method. The preliminary phenotypic detection of ESBLs was carried out by combination disc-diffusion test (CDDT) Performed using Kirby-bauer disc diffusion method on muller hinton agar. Results: Totally 10 clinical samples were collected and investigated Among 7 clinical samples of positive of Urinary tract infection (UTI), 20 micro-organisms belonging to 3 genus were isolated. Of the patients, 7 were female and 3 were male. E. coli and Klebsiella pneumonia were most prevalent species. E. coli and Klebsiella pneumonia were multidrug-resistant (resistance to five or more antibiotics) among the isolates. Of the isolated in Uro pathogens such as E. coli and Klebsiella pneumonia were ESBLs positive. Conclusion: Based on the results, E. coli and Klebsiella pneumonia predominated in the samples, giving the maximum frequency of ESBLs producing followed by E. coli (70%) and Klebsiella pneumonia (30%) from the combined disk diffusion method. Herbal medicine should be perform for UTI patients to prevent the spread of more. Only limited therapeutically options for ESBL-PE.