Efflux transporters have a considerable role in the multidrug resistance (MDR) of Pseudomonas aeruginosa, an important nosocomial pathogen. In this study, 45 P. aeruginosa clinical strains, with an MDR phenotype, have been isolated in a hospital of Northern Italy and characterized to identify the mechanisms responsible for their fluoroquinolone (FQ) resistance. These isolates were analyzed for clonal similarity, mutations in genes encoding the FQ targets, overexpression of specific Resistance Nodulation-cell Division efflux pumps, and search for mutations in their regulatory genes. The achieved results suggested that the mutations in genes encoding ciprofloxacin targets represented the main mechanism of FQ resistance of these strains; 97.8% of these isolates showed mutations in gyrA, 28.9% in gyrB, 88.9% in parC, and 6.7% in parE. Another mechanism of resistance was overexpression of the efflux pumps in some representative strains. In particular, overexpression of MexXY-OprM drug transporter was found in five isolates, whereas overexpression of MexCD-OprJ was detected in two isolates; surprisingly, in one of these last two isolates, also overexpression of MexAB-OprM pump was identified.