AbstractThere has been tremendous spread of antimicrobial resistance globally, mainly due to the excessive and unnecessary use of antibiotics, making the situation alarming. This has created a need for the development of alternative strategies to selectively target the bacterial pathogenicity without exerting selection pressure for the development of antimicrobial resistance. Targeting quorum sensing (QS)‐mediated virulence and biofilms by nontoxic natural products is gaining importance as new control strategy to combat the virulence and biofilms of pathogenic bacteria. In this study, the crude extract of Plumbago zeylanica was fractioned in different solvents using liquid–liquid partitioning to obtain the most bioactive fraction. The inhibitory effect of the bioactive extract of P. zeylanica on QS at sub‐minimum inhibitory concentrations (MICs) was studied against Chromobacterium violaceum 12472, Pseudomonas aeruginosa PAO1, and Serratia marcescens MTCC 97. Biofilm inhibition was studied using microtiter plate assay, scanning electron microscopy, and confocal laser scanning microscopy. Major phytocompounds detected were cinnamaldehyde dimethyl acetal, plumbagin, asarone, 4‐chromanol, phthalic acid, palmitic acid, ergost‐5‐en‐3‐ol, stigmasterol, and β‐sitosterol. The violacein production in C. violaceum 12472 was reduced by >80% in the presence of P. zeylanica hexane fraction (PZHF; 200 μg/ml). The most active PZHF inhibited QS‐mediated virulence factors of P. aeruginosa PAO1 such as pyocyanin, pyoverdin, rhamnolipid production, motility, etc., significantly at sub‐MICs. Similarly, PZHF showed 59 to 76% inhibition of biofilm formation of above test pathogens. The findings revealed that active fraction of P. zeylanica was effective against the QS‐regulated functions and biofilms development of Gram ‐ve pathogenic bacteria.