Staphylococcus aureus, a Gram-positive bacterium has been implicated in a plethora of human infections by virtue of its biofilm-forming ability. Inhibition in microbial biofilm formation has been found to be a promising approach towards compromising microbial pathogenesis. In this regard, various natural and synthetic molecules have been explored to attenuate microbial biofilm. In this study, the role of an amino acid, L-tryptophan was examined against the biofilm-forming ability of S. aureus. The compound did not execute any antimicrobial characteristics, instead, showed strong antibiofilm activity with the highest biofilm inhibition at a concentration of 50 µg/mL. Towards understanding the underlying mechanism of the same, efforts were given to examine whether tryptophan could inhibit biofilm formation by interfering with the quorum-sensing property of S. aureus. A molecular docking analysis revealed an efficient binding between the quorum-sensing protein, AgrA, and tryptophan. Moreover, the expression of the quorum-sensing gene (agrA) got significantly reduced under the influence of the test compound. These results indicated that tryptophan could interfere with the quorum-sensing property of the organism thereby inhibiting its biofilm formation. Further study revealed that tryptophan could also reduce the cell surface hydrophobicity of S. aureus by downregulating the expression of dltA. Moreover, the tested concentrations of tryptophan did not show any significant cytotoxicity. Hence, tryptophan could be recommended as a potential antibiofilm agent to manage the biofilm-associated infections caused by S. aureus.SUPPLEMENTARY INFORMATIONThe online version contains supplementary material available at 10.1007/s13205-021-02924-3.