Quercetin, a natural flavonoid, shows potential as a virulence inhibitor of Pseudomonas aeruginosa LasB. This study evaluated quercetin's impact on LasB production and activity in P. aeruginosa, including clinical strains. Molecular docking and dynamics simulations revealed favorable interactions between quercetin and LasB. At sub-MIC concentrations (≤256 μg/ml), quercetin effectively inhibited LasB production and activity, downregulated the lasB gene, and reduced the expression of QS system regulatory genes (lasI, lasR, rhlI, rhlR, pqsA, pqsR). Quercetin also competed with natural ligands (OdHDL, BHL, PQS) for binding to receptor proteins (LasR, RhlR, PqsR), forming more stable complexes. These findings suggest that quercetin disrupts the QS system, reducing LasB production and activity, offering an alternative for antivirulence therapy against P. aeruginosa infections. Quercetin exhibited significant inhibitory effects on LasB activity and gene expression in both PAO1 and clinical strains, with the most potent effect at 16 μg/ml. It also inhibited AHL synthesis, further supporting its role in disrupting QS. Quercetin's competitive binding to QS receptor proteins and its ability to form stable complexes with these proteins highlight its potential as an antivirulence agent. Overall, quercetin shows promise in combating LasB production and activity by targeting the QS system in P. aeruginosa.Quercetin, a natural flavonoid, shows potential as a virulence inhibitor of Pseudomonas aeruginosa LasB. This study evaluated quercetin's impact on LasB production and activity in P. aeruginosa, including clinical strains. Molecular docking and dynamics simulations revealed favorable interactions between quercetin and LasB. At sub-MIC concentrations (≤256 μg/ml), quercetin effectively inhibited LasB production and activity, downregulated the lasB gene, and reduced the expression of QS system regulatory genes (lasI, lasR, rhlI, rhlR, pqsA, pqsR). Quercetin also competed with natural ligands (OdHDL, BHL, PQS) for binding to receptor proteins (LasR, RhlR, PqsR), forming more stable complexes. These findings suggest that quercetin disrupts the QS system, reducing LasB production and activity, offering an alternative for antivirulence therapy against P. aeruginosa infections. Quercetin exhibited significant inhibitory effects on LasB activity and gene expression in both PAO1 and clinical strains, with the most potent effect at 16 μg/ml. It also inhibited AHL synthesis, further supporting its role in disrupting QS. Quercetin's competitive binding to QS receptor proteins and its ability to form stable complexes with these proteins highlight its potential as an antivirulence agent. Overall, quercetin shows promise in combating LasB production and activity by targeting the QS system in P. aeruginosa.