Syzygium cumini L. Skeels (Myretacae family) is a native plant of the Indian subcontinent which has wide socio-economical importance and is well known for its ant diabetic activity. The present study aimed to investigate the antibiofilm activity of purified fraction (EA) from S. cumini leaf extract against P. aeruginosa and S. aureus. The EA did not show any effect on growth of P. aeruginosa and S. aureus at the concentration of 900 µg/ml. At this concentration EA showed biofilm inhibition up to 86 ± 1.19% (*** P < 0.0001) and 86.40 ± 1.19% (*** P < 0.0001) in P. aeruginosa and S. aureus respectively. SEM examination also confirmed the reduction in biofilm formation. Further EA also disrupted some virulence phenotypes in P. aeruginosa and S. aureus. Bioactive compounds detected by GC–MS showed their possible molecular interaction with RhlG/NADP active-site complex (PDB ID: 2B4Q), LasR-TP4 complex (PDB ID: 3JPU) and Pseudaminidase (PDB ID: 2W38) from P. aeruginosa. The in vitro biofilm inhibition, virulence factor inhibition and the mode of interaction of bioactive components in Syzygium cumini with QS proteins of bacteria reported in this study might be an affordable and effective alternative method of controlling quorum sensing/biofilm-associated infections.