Aim: To determine the antimicrobial activity of costus (Saussurea lappa) oil against Staphylococcus aureus, and to evaluate the influence of subinhibitory concentrations of costus oil on virulence-related exoprotein production in staph. aureus. Methods and Results: Minimal inhibitory concentrations (MICs) were determined using a broth microdilution method, and the MICs of costus oil against 32 Staph. aureus strains ranged from 0.15 to 0.6 mu l ml-1. The MIC50 and MIC90 were 0.3 and 0.6 mu l ml-1, respectively. Western blot, haemolytic, tumour necrosis factor (TNF) release and real-time RT-PCR assays were performed to evaluate the effects of subinhibitory concentrations of costus oil on virulence-associated exoprotein production in Staph. aureus. The data presented here show that costus oil dose dependently decreased the production of alpha-toxin, toxic shock syndrome toxin 1 (TSST-1) and enterotoxins A and B in both methicillin-sensitive Staph. aureus (MSSA) and methicillin-resistant Staph. aureus (MRSA). Conclusion: Costus oil has potent antimicrobial activity against Staph. aureus, and the production of alpha-toxin, TSST-1 and enterotoxins A and B in Staph. aureus was decreased by costus oil. Significance and Impact of the Study: The data suggest that costus oil may deserve further investigation for its potential therapeutic value in treating Staph. aureus infections. Furthermore, costus oil could be rationally applied in food products as a novel food preservative both to inhibit the growth of Staph. aureus and to repress the production of exotoxins, particularly staphylococcal enterotoxins.