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Aim: The aim of this study was to detect prevalence of Extended Spectrum Beta Lactamase (ESBL) among Enterobacteriaceae isolated from various clinical samples received in a tertiary care hospital of Mumbai. Materials and Methods: This study was a retrospective study of 13 months (April 2017 to April 2018) done in the section of Microbiology of our institute. All gram negative isolates were identified and their antibiotic sensitivity pattern was studied according to standard microbiological procedures. ESBL activity among Enterbacteriaceae class of bacteria were confirmed by phenotypic confirmatory disc diffusion test according to Clinical Laboratory Standards Institute guidelines (CLSI) 2017. Clinical and demographic data was collected from Hospital Information and Management system (HIMS). Result: Total Enterobacteriaceae isolates studied were 1194 out of which 32.7% isolates were ESBL producers. ESBL producing Escherichia coli (E.coli) accounted to 34.8% of the total E.coli isolates and 30.7% of total Klebsiella pneumoniae (K.pneumoniae) isolates were ESBL producers. ESBL producing Klebsiella oxytoca (K.oxytoca) and Proteus mirabilis (P.mirabilis) were 29.7% and 18.6% respectively. Conclusion: Knowledge of prevalence of ESBL isolates in our institute will play a pivotal role in curtailing the use of unnecessary antibiotics and assist in taking measures to prevent their spread. Keywords: Extended spectrum beta lactamase, Enterobacteriaceae, Multidrug resistance.