Aim: To determine the prevalence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Gram-negative bacterial isolates from various clinical specimens submitted to a tertiary care hospital microbiology laboratory. Material and Methods: This hospital-based, cross-sectional study was conducted over a period of six months in the Department of Microbiology. A total of 140 non-duplicate Gram-negative isolates were obtained from clinical specimens including urine, blood, sputum, endotracheal aspirates, pus, wound swabs, and body fluids. Standard microbiological techniques were used for isolation and identification. Antimicrobial susceptibility testing was performed using the modified Kirby-Bauer disc diffusion method following Clinical and Laboratory Standards Institute (CLSI) guidelines. Results: Out of 140 isolates, the majority were obtained from urine samples (41.43%), followed by pus/wound swabs (22.86%) and sputum (12.86%). The most frequently isolated organisms were Escherichia coli (37.14%), Klebsiella pneumoniae (25.71%), and Pseudomonas aeruginosa (17.14%). MDR was observed in 78 isolates (55.71%), while XDR was identified in 28 isolates (20.00%). Among MDR organisms, E. coli (38.46%) and K. pneumoniae (28.21%) predominated. In the XDR group, A. baumannii (35.71%) was the leading isolate. High resistance rates were seen for ceftriaxone (84.62%), ciprofloxacin (79.49%), and cefepime (76.92%) among MDR isolates. In XDR isolates, resistance to imipenem and meropenem was 85.71% and 92.86%, respectively. Alarmingly, colistin resistance was noted in 42.86% of XDR isolates. Conclusion: There is a significant burden of MDR and XDR Gram-negative bacilli in clinical infections, particularly those caused by P. aeruginosa and A. baumannii. The alarming resistance to last-resort antibiotics such as carbapenems and colistin necessitates urgent implementation of antimicrobial stewardship, infection control strategies, and periodic resistance surveillance to guide empirical therapy effectively.