Quercetin, a natural flavonoid, has shown promising potential as an anti-virulence agent against *Pseudomonas aeruginosa*. The study evaluated the impact of quercetin on *P. aeruginosa* LasB, a key virulence factor, and elucidated the underlying mechanisms. Molecular docking and molecular dynamics simulations revealed favorable interactions between quercetin and LasB. At sub-MICs (≤256 μg/ml), quercetin effectively inhibited the production and activity of LasB elastase and downregulated the *lasB* gene transcription in both PAO1 and clinical *P. aeruginosa* strains. Correlation analysis showed significant positive correlations between the *lasB* gene and QS system regulatory genes (*lasI*, *lasR*, *rhlI*, and *rhlR*) in clinical strains. Quercetin also significantly downregulated the expression levels of these regulatory genes and competed with natural ligands for binding to receptor proteins (LasR, RhlR, and PqsR), disrupting the QS system. These findings suggest that quercetin has the potential to combat *P. aeruginosa* infections by inhibiting LasB production and activity through disruption of the QS system.Quercetin, a natural flavonoid, has shown promising potential as an anti-virulence agent against *Pseudomonas aeruginosa*. The study evaluated the impact of quercetin on *P. aeruginosa* LasB, a key virulence factor, and elucidated the underlying mechanisms. Molecular docking and molecular dynamics simulations revealed favorable interactions between quercetin and LasB. At sub-MICs (≤256 μg/ml), quercetin effectively inhibited the production and activity of LasB elastase and downregulated the *lasB* gene transcription in both PAO1 and clinical *P. aeruginosa* strains. Correlation analysis showed significant positive correlations between the *lasB* gene and QS system regulatory genes (*lasI*, *lasR*, *rhlI*, and *rhlR*) in clinical strains. Quercetin also significantly downregulated the expression levels of these regulatory genes and competed with natural ligands for binding to receptor proteins (LasR, RhlR, and PqsR), disrupting the QS system. These findings suggest that quercetin has the potential to combat *P. aeruginosa* infections by inhibiting LasB production and activity through disruption of the QS system.