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Biofilms play a decisive role in the infectious process and the development of antibiotic resistance. The establishment of bacterial biofilms is regulated by a signal-mediated cell–cell communication process called “quorum sensing” (QS). The identification of quorum sensing inhibitors (QSI) to mitigate the QS process may facilitate the development of novel treatment strategies for biofilm-based infections. In this study, the traditional medicinal plant <i>Ocimum sanctum</i> was screened for QS inhibitory potential. Sub-MICs of the extract significantly affected the secretion of EPS in Gram-negative human pathogens such as <i>Escherichia coli</i>, <i>Pseudomonas aeruginosa</i> PAO1, <i>Proteus mirabilis</i>, and <i>Serratia marcescens</i>, as well as aquaculture pathogens <i>Vibrio harveyi</i>, <i>V. parahaemolyticus</i>, and <i>V. vulnificus</i>, which render the bacteria more sensitive, leading to a loss of bacterial biomass from the substratum. The observed inhibitory activity of the <i>O. sanctum</i> extract might be attributed to the presence of eugenol, as evidenced through ultraviolet (UV)-visible, gas chromatography-mass spectroscopy (GC–MS), Fourier transformer infrared (FTIR) spectroscopy analyses, and computational studies. Additionally, the QSI potential of eugenol was corroborated through in vitro studies using the marker strain <i>Chromobacterium violaceum</i>.