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Abstract Background Biofilm formation is regarded as a significant factor in the establishment of infections caused by Pseudomonas aeruginosa. P. aeruginosa is one of the most important causes of nosocomial infections. Today silver nanoparticles (Ag-NPs) are used as antimicrobials due to their well-known, chemical, biological, and physical properties. Exposure to nanoparticles could inhibit colonization of new bacteria onto the biofilm. Methods In the present work, the green synthesis of Ag-NPs was performed using the alcoholic extract of Eucalyptus camaldulensis. Ag-NPs and glutathione-stabilized silver nanoparticles (GSH–Ag-NPs) were characterized using X-ray diffraction (XRD), dynamic light scattering (DLS), scanning electron microscope (SEM), and carbon, nitrogen, and hydrogen (CNH) and Fourier transform infrared spectroscopy (FTIR) techniques were applied to investigate the structure of the modified nanoparticles. Then, the antimicrobial and antibiofilm potential of the prepared Ag-NPs and GSH–Ag-NPs against P. aeruginosa strains was evaluated using microbroth dilution method and their effects on the expression of las I and las R genes. Results In this study, a total of 50 P. aeruginosa isolates were recovered from clinical samples. According to the results, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) value of Ag-NPs against P. aeruginosa was determined to be 512–256 μg/ml, respectively, while the MIC and MBC value of GS–Ag-NPs against P. aeruginosa clinical strains was determined in a range of 128–256 μg/ml and 256–512 μg/ml, respectively. The mean expression level in las R, las I genes in P. aeruginosa strains treated with ½ MIC of Ag-NPs was decreased by −5.7 and −8fold, respectively. The mean expression levels of las R, las I genes in P. aeruginosa strains treated with ½ MIC of GS–Ag-NPs were decreased by −8.7 and −10fold, respectively (P