Staphylococcus aureus is a serious foodborne pathogen threatening food safety and public health. Especially the emergence of methicillin-resistant Staphylococcus aureus (MRSA) increased the difficulty of S. aureus treatment. Staphyloxanthin is a crucial virulence factor of S. aureus. Blocking staphyloxanthin production could help the host immune system counteract the invading S. aureus cells. In this study, we first screened for staphyloxanthin inhibitors using a virtual screening method. The outcome of the virtual screening method resulted in the identification of eugenol (300 μg/mL), which significantly inhibits the staphyloxanthin production in S. aureus ATCC 29213, S. aureus Newman, MRSA ATCC 43300 and MRSA ATCC BAA1717 by 84.2%, 63.5%, 68.1%, and 79.5%, respectively. The outcome of the growth curve assay, field-emission scanning electron, and confocal laser scanning microscopy analyses confirmed that eugenol at the test concentration did not affect the morphology and growth of S. aureus. Moreover, the survival rate of S. aureus ATCC 29213 and MRSA ATCC 43300 under H2O2 pressure decreased to 51.9% and 45.5% in the presence of eugenol, respectively. The quantitative RT-PCR and molecular simulation studies revealed that eugenol targets staphyloxanthin biosynthesis by downregulating the transcription of the crtM gene and inhibiting the activity of the CrtM enzyme. Taken together, we first determined that eugenol was a prominent compound for staphyloxanthin inhibitor to combat S. aureus especially MRSA infections.