Enterococcus faecalis fsr quorum sensing (QS) involves an 11-residue cyclic peptide named gelatinase biosynthesis-activating pheromone (GBAP) that autoinduces two pathogenicity-related extracellular proteases in a cell density-dependent fashion. To identify anti-pathogenic agents that target fsr QS signaling, peptide antagonists of GBAP were created by our unique drug design approach based on reverse alanine scanning. First of all, a receptor-binding scaffold (RBS), [Ala(4,5,6,8,9,11)]Z-GBAP, was created, in which all amino acids within the ring region of GBAP, except for two essential aromatic residues, were substituted to alanine. Next, the substituted alanine residues were changed back to the original amino acid one by one, permitting selection of those peptide combinations exhibiting increased antagonist activity. After three cycles of this reverse alanine scan, [Ala(5,9,11)]Z-GBAP was obtained as a maximally reverted peptide (MRP) holding the strongest antagonist activity. Then, the fifth residue in MRP, which is one of the critical residues to determine agonist/antagonist activity, was further modified by substituting with different types of amino acids including unnatural amino acids. As a result, [Tyr(Bzl)(5), Ala(9,11)]Z-GBAP, named ZBzl-YAA5911, showed the strongest antagonist activity [IC(50) = 26.2 nM and Kd against GBAP receptor (FsrC) = 39.4 nM]. In vivo efficacy of this peptide was assessed with an aphakic rabbit endophthalmitis model. ZBzl-YAA5911 suppressed the translocation of E. faecalis from the aqueous humor into the vitreous cavity by more than 1 order of magnitude and significantly reduced retinal damage. We propose that ZBzl-YAA5911 or its derivatives would be useful as anti-infective agents to attenuate virulence expression in this opportunistic pathogen.