Multidrug resistance (MDR) in pathogenic bacteria has emerged as a significant clinical challenge. Targeting quorum sensing (QS), which regulates bacterial virulence, represents a promising strategy for combating MDR infections. In this study, we aimed to evaluate the anti-quorum sensing potential of selected medicinal plants against bacterial pathogens, alongside in silico interactions of their bioactive phytocompounds with QS and biofilm-associated proteins. Based on ethnopharmacological relevance, 18 plants were selected, and methanolic extracts were tested against Chromobacterium violaceum 12472. Among them, Acacia nilotica exhibited the highest activity and was further fractionated using solvents of increasing polarity (n-hexane, chloroform, and ethyl acetate) to assess anti-QS activity. The ethyl acetate fraction, identified as the most active, was evaluated at sub-MIC levels for its effect on QS-regulated virulence factors in Pseudomonas aeruginosa PAO1 and Serratia marcescens MTCC 97. Biofilm inhibition was quantified using microtiter plate assays and visualized through light microscopy. Phytochemical profiling of the ethyl acetate fraction was performed using GC/MS and LC/MS analyses. Molecular docking studies were conducted with AutoDock Vina to investigate interactions of the identified phytocompounds with QS proteins (LasI, LasR, CviR, RhlR) and biofilm-associated proteins (PilY1 and PilT). The MIC of the ethyl acetate fraction was determined as 250, 500, and 1000 μg/ml against C. violaceum 12472, P. aeruginosa PAO1, and S. marcescens MTCC97, respectively. At sub-MIC concentrations, the fraction significantly inhibited QS-regulated virulence factors and reduced biofilm formation by over 50%. GC/MS analysis revealed benzoic acid, 3,4,5-trihydroxy-, methyl ester (61.24%) as the major bioactive compound, while LC-MS detected retronecine in A. nilotica pods for the first time. In silico analysis showed that dehydroabietic acid occupied the CviR ligand-binding domain similarly to its antagonist, while betulin and epicatechin gallate interacted with PilY1 and PilT, disrupting biofilm formation. Overall, the findings indicate that phytochemicals from A. nilotica pods hold potential as anti-QS agents against Gram-negative pathogens. Further studies are warranted to evaluate the therapeutic efficacy of standardized extracts or isolated compounds in vivo.
