Multidrug resistance in hospital-acquired Pseudomonas aeruginosa isolates poses significant challenges in patient treatment. Understanding the molecular mechanisms underlying antibiotic resistance in P. aeruginosa infections is crucial for developing effective therapeutic strategies. This study aimed to investigate the antibiotic resistance patterns and the expression levels of the efflux pump genes MexCD-OprJ and MexEF-OprN in resistant P. aeruginosa isolates collected from clinical samples in Mashhad. A total of 102 P. aeruginosa isolates were identified using phenotypic and biochemical methods. Antibiotic susceptibility was assessed by the agar disk diffusion method using cefepime, aztreonam, norfloxacin, levofloxacin, ofloxacin, ceftiofur, and ceftriaxone disks. The minimum inhibitory concentration (MIC) for levofloxacin was determined by agar dilution method. Expression levels of mexC and mexE genes in levofloxacin-resistant isolates were evaluated using real-time PCR.The results showed that all isolates were resistant to ceftriaxone. Resistance rates to ceftiofur, cefepime, and aztreonam were 93%, 65%, and 54.4%, respectively. Among the fluoroquinolones (norfloxacin, levofloxacin, and ofloxacin), the overall resistance rate was approximately 71%. Multidrug resistance was observed in 53.4% of the isolates. The highest proportion of isolates (22.4%) exhibited an MIC of 128 µg/ml for levofloxacin. Overexpression of mexE and mexC was statistically significant in 11 (55%) and 9 (45%) of the resistant isolates, respectively. However, in the remaining isolates, changes in gene expression—whether increased or decreased was not statistically significant, suggesting the involvement of alternative resistance mechanisms. Overall, efflux pump overexpression appears to be a key contributor to antibiotic resistance in these clinical isolates.