ABSTRACTAntibiotic tolerance within a biofilm community presents a serious public health challenge. Here, we report the identification of a 2-aminoimidazole derivative that inhibits biofilm formation by two pathogenic Gram-positive bacteria,Streptococcus mutansandStaphylococcus aureus. InS. mutans, the compound binds to VicR, a key response regulator, at the N-terminal receiver domain, and concurrently inhibits expression ofvicRand VicR-regulated genes, including the genes that encode the key biofilm matrix producing enzymes, Gtfs. The compound inhibitsS. aureusbiofilm formation via binding to a Staphylococcal VicR homolog. In addition, the inhibitor effectively attenuatesS. mutansvirulence in a rat model of dental caries. As the compound targets bacterial biofilms and virulence through a conserved transcriptional factor, it represents a promising new class of anti-infective agents that can be explored to prevent or treat a host of bacterial infections.