The antibacterial activities of vulgarone B, a component of Artemisia iwayomogi essential oil, were evaluated against some antibiotic-susceptible and -resistant human pathogens. Moreover, the effects of combining antibiotics, such as oxacillin, with vulgarone B were determined in this study. Significant inhibitory activities of Artemisia oils against antibiotic-susceptible and -resistant bacteria were confirmed by broth microdilution methods. The effects of vulgarone B on bacterial morphology and DNA were observed by scanning electron microscope and electrophoresis, respectively. In checkerboard microtiter tests, vulgarone B and A. iwayomogi oil combined with oxacillin resulted in synergism, or additive effects. Moreover, the safety of Artemisia oil and vulgarone B were confirmed in vivo. Both vulgarone B and the essential oil fraction of A. iwayomogi showed significant inhibitory activities against strains of antibiotic-susceptible and -resistant bacteria. The oils showed synergism or additive effects when combined with oxacillin against two strains of Staphylococcus aureus. The antibiotic mechanism involved might be related to DNA cleavage. Thus, vulgarone B and the essential oil fraction of A. iwayomogi may be promising candidates for a safe, effective, natural agent active against antibiotic-resistant S. aureus, especially when combined with oxacillin.