In bacteria, quorum sensing (QS) is a process of chemical communication involving the production, release, and subsequent detection of signaling molecules. QS regulates the production of key virulence factors in pathogens. During the screening of herbal extracts, clove extract was found to inhibit QS-controlled gene expression in Pseudomonas aeruginosa QSIS-lasI and Chromobacterium violaceum CV026 biosensors. Using a bioautographic TLC assay, preparative TLC, and HPLC analysis, eugenol, the major constituent of clove extract, exhibited QS inhibitory activity. Eugenol at sub-inhibitory concentrations inhibited the production of virulence factors, including violacein, elastase, pyocyanin, and biofilm formation. Using two Escherichia coli biosensors, MG4/pKDT17 and pEAL08-2, we confirmed that eugenol inhibited the las and pqs QS systems. Our data identified eugenol as a novel QS inhibitor. The purpose of this study was to track the quorum sensing inhibitor (QSI) in herbal extracts by effective screening systems and evaluate its biological activity. The QSIs from herbal extracts are potential agents for the treatment of bacterial infections.