Objective: The emergence of antibiotic resistance in Pseudomonas aeruginosa and Acinetobacter baumanii isolates poses serious risks to public health. Our study aimed to investigate the presence of colistin resistance and mcr-1 gene positivity in these isolates. Material and Method: Culture, biochemical tests, antibiotic susceptibility tests, and molecular tests were used to isolate and identify P. aeruginosa and A. baumanii strains. Result and Discussion: A sum of 156 clinical isolates of Pseudomonas aeruginosa (n = 89) and Acinetobacter baumannii (n = 67) were obtained using the 550 clinical samples collected in one year from the largest hospital in Van, Turkey. The results of antibiotic susceptibility tests showed that approximately 82.8% of P. aeruginosa and 94.6% of A. baumannii strains were multidrug-resistant (MDR). Colistin resistance was detected in 11.23% (10/89) of P. aeruginosa isolates and 11.94% (8/67) of A. baumannii isolates using agar dilution and microdilution methods. Out of the 18 colistin-resistant isolates, the mcr-1 gene was detected in three P. aeruginosa and two A. baumannii strains. The detection of plasmid-mediated colistin resistance in P. aeruginosa and A. baumannii is of great concern due to the high potential for colistin resistance to spread in clinical settings. Understanding the unique circumstances of worldwide colistin resistance can be facilitated by promoting the creation of quick processes for identifying colistin resistance profiles and putting them into practice in hospital laboratories. Colistin and carbapenem treatment are two effective ways to treat emerging resistant super-microbes and slow down the emergence of resistance.