Introduction: The major resistance mechanisms of Pseudomonas aeruginosa to fluoroquinolones and carbapenems are associated with the mutations in the genes gyrA and oprD encoding type II topoisomerases (DNA gyrase) and OprD porin, respectively. Method: In this cross-sectional study, sixty five clinical samples were collected from patients hospitalized in Al-Zahra Hospital of Isfahan, Iran. Susceptibility testing was performed by using disk diffusion and minimum inhibitory concentration (MIC) by E-test methods as recommended by Clinical Laboratory Standards Institute (CLSI). The assay was based on a DNA sequencing method using polymerase chain reaction (PCR). Results: The disk diffusion and E-test methods showed significant concordance in determining the in-vitro activity of the meropenem and ciprofloxacin against P. aeruginosa isolates. The mutations associated with antibiotic resistance were detected in the codon 83 of the gyrA gene, and various codons of the oprD gene. Conclusion: Our results showed that the main mechanism of fluoroquinolone resistance in P. aeruginosa is mediated primarily through mutations in gyrA and carbapenem resistance was driven mainly by the mutational inactivation of oprD gene.