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Introduction: Antimicrobial resistance is increasing and becoming a major public health problem worldwide. The continued emergence of multidrug-resistant (MDR) and extended-spectrum beta-lactamases (ESBL) producing Enterobacteriaceae strains lead to the higher rate of morbidity and mortality among hospitalized patients. Obtaining regional drug resistance data is important for developing guidelines for appropriate antibiotic use and to control the emergence of antibiotic resistance. In this background this study aims to determine the prevalence of MDR and ESBL producing Gram Negative Enterobacteriaceae. Materials and Methods: The Present cross-sectional study was carried out in Department of Microbiology, P.D.U. medical college, Rajkot between February 2024 and June 2024. A total of 900 Gram-negative Enterobacteriaceae isolates from various clinical specimens were subjected to antibiotic susceptibility testing as per the Clinical and Laboratory Standards Institute guidelines. Results: Out of the total 900 Enterobacteriaceae isolates, ESBLs production was confirmed in 341 isolates with isolation rate of 37.87%. Out of these maximum isolates were Escherichia coli (43.18%) followed by Klebsiella pneumoniae (38.77%). Maximum number of cases 169 (49.56%) were observed in older age group. In our study, 522 (58%) isolates were found to be MDR isolates. Antibiotic sensitivity of ESBL producers was observed highest to Meropenem (100%) and Amikacin (84.75%). Conclusion: Isolation rate of MDR and ESBL producing Enterobacteriaceae strains was higher in hospitalized patients which indicates these isolates are vital in health care associated infections and requires proper infection control measures. These isolates have high rate of resistance against commonly used drugs of antimicrobial categories like cephalosporins and fluoroquinolones which indicates to formulate strict antibiotics prescription policy in health care settings.