Eugenol, an essential oil component in plants, has been demonstrated to possess activity against both Gram-positive and Gram-negative bacteria. This study examined the influence that subinhibitory concentrations of eugenol may have on the expression of the major exotoxins produced by Staphylococcus aureus. The results from a tumor necrosis factor (TNF) release assay and a hemolysin assay indicated that S. aureus cultured with graded subinhibitory concentrations of eugenol (16 to 128 g/ml) dose dependently decreased the TNF-inducing and hemolytic activities of culture supernatants. Western blot analysis showed that eugenol significantly reduced the production of staphylococcal enterotoxin A (SEA), SEB, and toxic shock syndrome toxin 1 (the key exotoxins to induce TNF release), as well as the expression of ␣-hemolysin (the major hemolysin to cause hemolysis). In addition, this suppression was also evaluated at the transcriptional level via real-time reverse transcription (RT)-PCR analysis. The transcriptional analysis indicated that 128 g/ml of eugenol remarkably repressed the transcription of the S. aureus sea, seb, tst, and hla genes. According to these results, eugenol has the potential to be rationally applied on food products as a novel food antimicrobial agent both to inhibit the growth of bacteria and to suppress the production of exotoxins by S. aureus.Staphylococcus aureus is a major nosocomial pathogen. This species is capable of causing a wide range of diseases and is often associated with high morbidity and mortality rates. The disease spectrum includes food-borne illness, cutaneous infections, endocarditis, pneumonia, septic arthritis, and osteomyelitis (24, 36). The diversity is, in part, dependent on the secretion of a broad spectrum of soluble extracellular proteins. These proteins include enterotoxins, hemolysins, toxic shock syndrome toxin 1 (TSST-1), and others (20). S. aureus infections are difficult to control due to a combination of toxinmediated virulence, invasiveness, and antibiotic resistance. Therefore, there is a continuing need to discover new and improved antimicrobial agents to treat S. aureus illnesses, with potential benefits for both the food and pharmaceutical industries (35).Enterotoxins, TSST-1, and ␣-hemolysin are among the major exotoxins secreted by S. aureus. Staphylococcal enterotoxins (SEs) are a group of major serological heat-stable enterotoxins (SEA to SEE and SEG to SEJ) (3). These toxins cause staphylococcal gastroenteritis, food poisoning, toxic shock-like syndromes, and several allergic and autoimmune diseases (3,22,35). TSST-1 is the key causative toxin of toxic shock syndrome (TSS), which is an acute-onset and potentially lifethreatening staphylococcal syndrome (23). Furthermore, SEs and TSST-1 are also known to be pyrogenic toxin superantigens (PTSAgs) that stimulate the proliferation of T lymphocytes and the release of T-cell-derived cytokines (12). ␣-Hemolysin is a 33-kDa pore-forming soluble protein that has hemolytic, cytolytic, and dermonecrotic activities....