The present study was carried out to assess the anti-quorum sensing (anti-QS) activity of bark extract obtained from the mangrove plant Rhizophora annamalayana Kathir. against Gram-negative bacteria. In microtitre plate assay, the bark extract at a concentration of 1 mg/ml inhibited the QS-dependent violacein production in Chromobacterium violaceum ATCC 12472. Further, the QS-dependent bioluminescence production in the aquatic bacterial pathogen Vibrio harveyi MTCC 3438 was also reduced to the level of 99 % when treated with the same concentration of the extract. Gas chromatography-mass spectrum analysis identified the presence of seven different chemical constituents, 1H-purin-6-amine, cycloheptasiloxane, cyclooctasiloxane, cyclononasiloxane, cyclononasiloxane octadecamethyl, cyclodecasiloxane eicosamethyl and 1,1,1,5,7,7,7-heptamethyl-3,3-bis(trimethylsiloxy)tetrasiloxane. The molecular docking analysis of the identified compounds revealed that the compounds cyclononasiloxane octadecamethyl and cyclodecasiloxane eicosamethyl exhibited the best docking energy with the QS receptors of C. violaceum and V. harveyi with that of the natural ligand N -hexanoyl- L -homoserine lactone (C6-HSL) and furanosyl borate diester (AI-2). Similarly, another compound 1,1,1,5,7,7,7-heptamethyl-3,3-bis(trimethylsiloxy)tetrasiloxane showed best docking energy only against C6-HSL. Thus, the results of the present study divulge the activity of R. annamalayana bark extract to interfere with bacterial QS.