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The potential of soybean isoflavones (SI) as a quorum sensing (QS) inhibitor against Pseudomonas aeruginosa was investigated in the present study. The inhibitory effects of SI on violacein production, biofilm formation, motility, and virulence factor production were evaluated. The acyl-homoserine lactones (AHLs) were quantified by liquid chromatography-mass spectrometry (LC-MS). The QS-related genes were analyzed by qRT-PCR. The molecular docking analysis was employed to investigate the anti-QS mechanism of the SI. Results demonstrated that the SI exhibited significant inhibition (p < 0.05) in violacein production, biofilm formation, motility, virulence factors, AHLs production, and expression levels of QS-related genes in P. aeruginosa. Additionally, the result of molecular docking analysis revealed that SI had a promising competitive interaction with AHLs. Hence, the study provided a deep insight into SI …