Quorum sensing is a cell-density-dependent regulatory system in gram-positive bacteria and is often regulated by cyclic peptides called "quormones," which function as extracellular communication signals. With an aim to discover an antipathogenic agent targeting quorum sensing in gram-positive bacteria, we screened 153 samples of fungal butanol extracts with the guidance of the inhibition of quorum-sensing-mediated gelatinase production in Enterococcus faecalis. Following the screenings, we found that ambuic acid, a known secondary fungal metabolite, inhibited the quorum-sensing-mediated gelatinase production without influencing the growth of E. faecalis. We further demonstrated that ambuic acid targeted the biosynthesis of a cyclic peptide quormone called gelatinase biosynthesis-activating pheromone. Furthermore, ambuic acid also inhibited the biosynthesis of the cyclic peptide quormones of Staphylococcus aureus and Listeria innocua. These results suggest the potential use of ambuic acid as a lead compound of antipathogenic drugs that target the quorum-sensing-mediated virulence expression of gram-positive bacteria.