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 bacterial epistatic assays to elucidate the mode of action. We report the discovery and medicinal chemistry development of a substituted pyrazoloquinoline (ML370) shown to be an inhibitor of Vibrio cholerae LuxO, a response regulator and intracellular kinase. The probe acts directly on LuxO by inhibiting the ATPase activity. ML370 should greatly expand the general understanding of how QS response regulators relay information from upstream signals that lead to modified gene expression. In addition ML370 and compound analogues could lead to the development of antibacterial drugs designed to interfere with QS that could have enormous ramifications for improving human health.