Staphylococcus aureus is a significant cause of morbidity and mortality worldwide in both hospital- and community-acquired infections. Of additional serious concern, the pathogens are resistant to most available antibiotics. Many studies tried to discover new antimicrobial agents to reduce and prevent the antibiotic resistance problem. Rhodomyrtone, a member of acylphloroglucinols isolated from Rhodomyrtus tomentosa (Aiton) Hassk. leaves, exhibited pronounced antibacterial activity against a wide range of Gram-positive bacteria. However, antibacterial mechanisms of the compound against pathogenic bacteria have not been extensively studied. Therefore, the aim of this research was to investigate antibacterial mechanisms of the active compound against S. aureus ATCC 29213. Localization of rhodomyrtone in the pathogen was performed. The results demonstrated that the compound was detected in cell wall and cell membrane of S. aureus cells after treatment with 8 µg/ml of the active compound for 1-4 h. Moreover, rhodomyrtone penetrated into cytoplasm of the bacterial cells. Thus, a fine bane of the bioactive compound was observed in the cytoplasmic fraction of rhodomyrtone-treated S. aureus. In addition, rhodomyrtone elucidated a variety of effects on the properties and functions of macromolecular biosynthesis in S. aureus. Rhodomyrtone at sub-minimum inhibitory concentration (sub-MIC = 0.25 µg/ml) completely inhibited DNA synthesis within 30 min. Similar result was observed for incorporation of [³H]leucine into protein synthesis. For cell wall and lipid synthesis, inhibition of the pathways became evident after 60 min of the compound addition. In contrast, RNA synthesis pathway slightly decreased in the presence of rhodomyrtone. In all cases there was no loss in the amount of radioactive label from TCA-precipitable material after incubation up to 6 h. It might be no breakdown of labelled precursors. Thus, effects of rhodomyrtone on DNA synthesis in the pathogen will be further extensively studied.