Background: This research aimed to study the prevalence and emergence of high drug-resistant bacterial isolates from clinical samples in a tertiary care hospital and their antibiogram profile. The existence of multidrug-resistant organisms is on the rise across the globe and is a severe problem. Knowledge of the prevalence and antibiogram profile of such isolates is essential to develop an appropriate treatment methodology. Methods: A cross-sectional study was conducted in the Department of Microbiology, RMMCH, from July 2022 to December 2022. Different clinical specimens were collected from patients who were suspected of infections. A total of 470 clinical samples were collected and identified using conventional microbiological, biochemical tests following the Clinical and Laboratory Standard Institute (CLSI) guidelines. Antimicrobial susceptibility testing (AST) was performed using the in vitro Kirby-Bauer disk diffusion method. The screening test for ESBL producers was performed phenotypically using the double disk diffusion method. Results: Overall, 316 bacterial isolates were isolated from 470 patients who had visited the hospital. Of these, 263 (83.2%) were gram-negative and the remaining 53 (16.8%) were gram-positive. The most common isolates were Escherichia coli 84 (26.6%), Klebsiella pneumoniae 73 (23.1%), Staphylococcus aureus 53 (16.8%) and Pseudomonas aeruginosa 42 (13.3%). The overall rate of MDR, XDR and PDR bacterial isolates from RMMCH were found to be 89 (28.16%), 157 (49.68%), and 33 (10.44%) respectively. Highest MDR 23 (25.84%), XDR 55 (35.03%), and PDR 7 (21.21%) showed E.coli Prevalence of ESBL, AmpC, and Carbapenemase-producing isolates. Escherichia coli and Klebsiella pneumoniae were the predominant isolates and were also the major ESBL producers. Besides Clindamycin, Erythromycin, Polymyxin B, Piperacillin/Tazobactam, Meropenem and Imipenem showed high efficacy. Conclusions: In this study, a high