Antibiotic resistance is a major clinical and public health problem. Non-steroidal anti-inflammatory drugs (NSAIDs) could increase the susceptibility of bacteria to antibiotics and have shown broad antimicrobial activity. In this work, the effect of Diclofenac sodium alone and in combination with Gentamicin on the expression of efflux pump genes and some virulence traits in clinical isolates of P. aeruginosa was investigated. The checkerboard titration assay was used to evaluate the synergistic effect of Diclofenac sodium and Gentamicin. The relative expression of MexAB-OprM and MexXY-OprM efflux pump genes was determined using qPCR. The impact of drugs on the activity of the efflux pump and some virulence traits, including biofilm formation, swarming, swimming and twitching, and bacterial proteolytic and hemolytic activities were assessed. The minimum inhibitory concentration (MIC) of Diclofenac sodium and Gentamicin for clinical P. aeruginosa strains was 5120 and 128 µg/mL and the drugs showed synergic antibacterial activity. Diclofenac sodium reduced the expression of the mexB, mexX, and mexY genes, and increased the expression of the mexA and oprM genes. In addition, Diclofenac sodium alone and in combination with Gentamicin inhibited the activity of the bacterial efflux pump and the simultaneous treatment of P. aeruginosa with Diclofenac sodium and Gentamicin significantly reduced biofilm formation, bacterial motility, proteolytic (40.78%) and hemolytic (85%) activities compared with untreated group. This study addresses clinically relevant questions about the efficacy and potential synergistic effects of diclofenac sodium and gentamicin in treating P. aeruginosa infections that can be applicable to clinical practice.