BACKGROUND: Pseudomonas aeruginosa is a significant opportunistic pathogen, especially in hospital-acquired infections, with plasmid-mediated fluoroquinolone resistance posing a major healthcare threat. This research aimed to isolate fluoroquinolone-resistant P. aeruginosa from patients at Aleppo University Hospital, assess the prevalence of fluoroquinolone resistance, confirm molecular identity, identify plasmid-associated resistance genes, and investigate virulence factors. METHODS: A total of 430 samples were collected from patients and cultured on selective media for identification. Molecular confirmation was achieved through PCR techniques. Various media were used to assess virulence factors and antibiotic resistance while also investigating the prevalence of resistance-related genes. RESULTS: The study identified 29 fluoroquinolone-resistant P. aeruginosa isolates. These strains exhibited complete resistance to penicillins and all four generations of cephalosporins while remaining 100% sensitive to colistin. Notably, both hemolysin and gelatinase production rates were found to be 100%, and 48.2% of the isolates formed strong biofilms. The aac(6')-Ib gene was present in 72.4% of the isolates, the qnrS gene in 44.8%, and the qnrB gene in 13.7%. Additionally, 37.8% of the isolates contained two types of resistance genes, while 62% had one type. Importantly, all resistant isolates (100%) possessed at least four virulence factors. CONCLUSION: The findings indicate a prevalence of plasmid-associated fluoroquinolone resistance genes in the studied isolates. It is recommended to rationalize fluoroquinolone use to preserve their effectiveness against multidrug-resistant strains.