Quorum sensing (QS) is a process of bacterial cell-to-cell communication that relies upon recognition of extracellular signaling molecules called autoinducers. QS allows bacteria to synchronize their behavior in response to changes in the population density and species composition of the proximal bacterial community. Known behaviors regulated by QS include bioluminescence, sporulation, virulence factor production, and biofilm formation. We carried out a high throughput screen (HTS) to identify small molecules that modulate QS in a modified V. cholerae strain carrying a luciferase operon; activation of the quorum pathway is accompanied by light production. 352,083 compounds from the NIH-MLPCN compound library were evaluated. Potential QS modulators were characterized via additional bacterium-based epistatic assays to elucidate their mode of action. We report the discovery and development of two, structurally distinct, small-molecule probes (ML343 and ML344) shown to be agonists of Vibrio cholerae CqsS, a transmembrane receptor. ML343 and ML344 should greatly expand the general understanding of how QS membrane receptors productively interact with ligands and how they relay information from the external environment. In addition these compounds could lead to the development of antibacterial drugs designed to interfere with QS which could have enormous ramifications for improving human health.