Quorum sensing (QS) is a cell-cell communication mechanism by which bacteria synchronize social behaviors such as biofilm formation and virulence factor secretion by producing and sensing small molecular signals. Quorum quenching (QQ) by degrading signals or blocking signal transmissions has become a promising strategy for disrupting QS and preventing bacterial infection and biofilm formation. However, studies of high-throughput screening and identification approaches for quorum-sensing inhibitors (QSIs) are still inadequate. In this work, we developed a sensitive, high-throughput approach for screening QSIs based on the bacterial biosensor strain Agrobacterium tumefaciens N5 (pBA7P), which contains a traG gene promoter induced by QS signals fused with a promoterless β-lactamase gene reporter. Using this approach, we identified 31 QQ bacteria from ∼2000 soil bacterial isolates, some belonging to the genera Bosea, Cupriavidus, and Flavobacterium that have not been reported previously as QQ bacteria. We also identified four QS inhibitory compounds and one QS signal analogue from ∼5000 small-molecule compounds, which profoundly affected the expression of QS-regulated genes and phenotypes of the pathogenic bacteria. This high-throughput screening system is effective and sensitive for screening of both QQ microbes and small molecules, enabling the discovery of a wide variety of biocompatible compounds.