Abstract <p><b>Background</b>: <i>Pseudomonas aeruginosa</i> is a significant pathogen in nosocomial infections due to its adaptability and intrinsic resistance mechanisms. This study investigates the antibiotic and biocide susceptibility of <i>P. aeruginosa</i> isolates from clinical and hospital environments, emphasizing efflux pumpmediated resistance. <b>Materials and methods</b>: From November 2023 to April 2024 a total of 200 samples were collected, comprising 100 clinical and 100 environmental isolates from five hospitals in Hamadan, Iran. Antibiotic susceptibility was determined according to CLSI guidelines, and biocide susceptibility was tested against agents such as chlorhexidine and triclosan. Molecular analysis, including PCR and gene expression profiling, was performed to identify efflux pump-related resistance genes. <b>Results</b>: Among clinical isolates, 22% (22 isolates) were classified as multidrug-resistant (MDR), showing resistance to three or more antibiotic classes and among environmental isolates, 18% (18 isolates) were classified as multidrug-resistant (MDR). Biocide testing revealed reduced susceptibility to chlorhexidine and triclosan in clinical isolates compared to environmental isolates. Molecular analysis detected efflux pump-associated genes, including <i>qacE</i>, <i>qacE</i>Δ<i>1</i>, <i>mexA</i>, <i>mexX</i>, and <i>fabI</i> who <i>fabI</i> specifically linked to triclosan resistance. <b>Conclusions</b>: The findings demonstrate the complexity of <i>P. aeruginosa</i> resistance mechanisms, particularly efflux pump-mediated resistance. These results emphasize the necessity of stringent infection control practices and the development of innovative therapeutic strategies to combat this pathogen.</p>