Pseudomonas aeruginosa is a major causative agent of nosocomial and chronic infections, and its high resistance to antibiotics poses significant therapeutic challenges. One of the key mechanisms contributing to this resistance is the activity of multidrug efflux pumps, particularly MexAB-OprM and MexXY-OprM. This study aimed to investigate the expression of efflux pump genes mexA and mexX in multidrug-resistant (MDR) clinical isolates of P. aeruginosa. Clinical specimens were collected from patients at Imam Reza Hospital. Bacterial isolates were identified using standard culture and biochemical methods. Antibiotic susceptibility was evaluated against ceftazidime, chloramphenicol, piperacillin, colistin, and ceftazidime-clavulanic acid using disk diffusion, and minimum inhibitory concentrations (MICs) were determined via agar dilution. The expression levels of mexA and mexX genes were quantified using real-time PCR (qRT-PCR). Antibiotic susceptibility testing showed that all isolates were resistant to ceftazidime. Resistance to tetracycline and colistin was 96.88% and 93.75%, respectively. Resistance rates to ceftazidime-clavulanic acid, chloramphenicol, and piperacillin were 85.42%, 70.83%, and 67.71%, respectively. More than 84% of isolates exhibited high MIC values. Gene expression analysis revealed a significant upregulation of mexA and mexX in several MDR isolates. The observed positive correlation between efflux pump overexpression and multidrug resistance suggests that these systems play a critical role in reducing antibiotic efficacy by actively exporting drugs from the bacterial cell. In conclusion, the high prevalence of efflux pump overexpression in MDR P. aeruginosa highlights the importance of targeting these mechanisms in future therapeutic strategies. The alarming levels of resistance observed in this study underscore the urgent need for the development of novel and effective antimicrobial approaches.