ABSTRACTPolymicrobial intra-abdominal infections (IAI) involvingCandida albicansandStaphylococcus aureusare associated with severe morbidity and mortality (∼80%). Our laboratory discovered that the immunomodulatory eicosanoid prostaglandin E2(PGE2) plays a key role in the lethal inflammatory response during polymicrobial IAI using a mouse model of infection. In studies designed to uncover key PGE2biosynthesis/signaling components involved in the response, selective eicosanoid enzyme inhibitors and receptor antagonists were selected and prescreened for antimicrobial activity againstC. albicansorS. aureus. Unexpectedly, we found that the EP4receptor antagonist L-161,982 had direct growth-inhibitory effects onS. aureusin vitroat the physiological concentration required to block the PGE2interaction with EP4. This antimicrobial activity was observed with methicillin-sensitiveS. aureusand methicillin-resistantS. aureus(MRSA) strains, with the MIC and minimum bactericidal concentration values for planktonic cells being 50 μg/ml and 100 μg/ml, respectively. In addition, L-161,982 inhibitedS. aureusbiofilm formation and had activity against preformed mature biofilms. More importantly, treatment of mice with L-161,982 following intraperitoneal inoculation with a lethal dose of MRSA significantly reduced the bioburden and enhanced survival. Furthermore, L-161,982 protected mice against the synergistic lethality induced by coinfection withC. albicansandS. aureus. The antimicrobial activity of L-161,982 is independent of EP4receptor inhibitory activity; an alternative EP4receptor antagonist exerted no antimicrobial or protective effects. Taken together, these findings demonstrate that L-161,982 has potent antimicrobial activity against MRSA and may represent a significant therapeutic alternative in improving the prognosis of mono- or polymicrobial infections involving MRSA.