The LuxT regulator protein is a potential drug target for regulating quorum sensing (QS) in Vibrio alginolyticus. There is no substantiation of a 3D structure of LuxT in this particular species, thus the 3D model was constructed using molecular modeling and its stability was verified under solvation effect using molecular dynamics simulation. Further, exploration of the drug candidate against the LuxT binding site through structure-based approaches identified four Bitter compounds, viz., quercetin, cnicin, 5, 5′ methylenedisalicyclic acid, and flufenamic acid with high docking score and optimum binding energy. Remarkably, these compounds established agreeable interactions with the amino acid residues Phe67, Asp63, Thr59, Gly64, Ile66, Phe99, Arg123, Asn119, and Gly120; which are found to play a vital role in the LuxT mechanistic inhibition. In addition to the scoring and energy parameters, MD simulation and ADME prediction suggested that the proposed compounds may be promising drug candidates against V. alginolyticus. Therefore, the lead candidate quercetin was scrutinized to validate its potential in biofilm inhibition of V. alginolyticus. The results revealed that the compound had better efficacy in biofilm destruction; as a consequence, there was a significant effect on motility and EPS synthesis at an effective concentration of 100 μM, which was further confirmed by microscopic studies. Hence, the present study will provide an insight into the design of high potent drug molecules to combat dreadful pathogenic diseases caused by V. alginolyticus.