Background: Nosocomial infections caused by multi-drug resistant Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) are a growing public health concern. These bacteria, commonly found in healthcare settings, possess various resistance genes that enable them to evade multiple antibiotics, leading to increased morbidity and mortality. Understanding the prevalence and genetic mechanisms of these pathogens is essential for improving infection control and treatment strategies. Methods: This study collected 150 isolates of E. coli and K. pneumoniae from various clinical samples of hospitalized patients. The samples were cultured and incubated, and the isolates were identified and tested for antimicrobial sensitivity using both conventional and automated methods via the BD Phoenix M50 system. Resistant isolates were further analyzed for specific resistance genes through PCR to detect the presence of KPC, IMP, VIM, NDM, and OXA-48 genes, using the GeneXpert System. All procedures were conducted at the Armed Forces Labs for Medical Research and Blood Bank. Results: Out of 203 samples, 8 samples showed no growth, 150 isolates showed E. coli and K. pneumoniae, and 45 isolates showed other species. 23.3% of the 150 isolates were sensitive, while 76.7% contained MDR organisms. Among MDR, 65 out of 115 (56.5%) were ESBLs and 50 out of 115 (43.5%) were carbapenem-resistant. The majority of carbapenem-resistant isolates contained one antimicrobial resistance gene, 24% had two antimicrobial resistance genes, and only 6% had all three. The most common genes were NDM (66%), OXA-48 (58%), and KPC (12%). Conclusion: This study demonstrated that antimicrobial susceptibility testing revealed increased resistance to most antibiotics in K. pneumoniae isolates, particularly carbapenem resistance. In contrast, ESBL resistance was much greater in E. coli isolates. In addition, K. pneumoniae and E. coli-associated nosocomial infections contained an increased number of resistance genes.