Background and aim: Colistin and carbapenem-resistant Pseudomonas and Acinetobacter isolates can cause severe infections. We aimed to determine the prevalence of colistin and carbapenem resistance in Pseudomonas and Acinetobacter nosocomial isolates and investigate the underlying mechanisms. Methods: The antimicrobial susceptibility of Pseudomonas and Acinetobacter isolates was tested through disk diffusion method, while colistin resistance was tested by agar dilution method. Extended spectrum-β-lactamases (ESβLs) production was tested using the combined disk method, and carbapenemase production was tested phenotypically (using modified carbapenem inactivation method (mCIM) and Imipenem/EDTA combined disc diffusion test). Genotypic analysis detected carbapenemase genes. Colistin-resistant isolates were investigated for efflux pump mechanisms using the carbonyl cyanide 3-chlorophenylhydrazone (CCCP). Results: Fifty Pseudomonas and thirty Acinetobacter isolates were isolated from the collected samples. Approximately, 44% of Pseudomonas and 43.3% of Acinetobacter isolates produced ESβLs. Carbapenemase production was found in 38% of Pseudomonas and 40% of Acinetobacter isolates while 28% and 23.3% produced metallo-β-Lactamases (MβLs). Colistin resistance was detected in 14% of Pseudomonas and 10% of Acinetobacter isolates. CCCP reduced colistin MIC by ≥8 folds in 85.7% and 100% of colistin-resistant Pseudomonas and Acinetobacter isolates, respectively. The carbapenemase genes bla NDM, bla VIM-2 and bla IMP-1 were found in 33.3%, 16.7% and 6.7% of Pseudomonas isolates, and in 25%, 15% and 5% of Acinetobacter isolates, respectively. Conclusion: Pseudomonas and Acinetobacter isolates showed resistance to multiple antibiotics. Carbapenemase production shows challenges for effective treatment. Efflux pump inhibitors exhibited potential in reversing colistin resistance emphasizing the need to avoid unnecessary clinical use of colistin.