The antibacterial mechanisms of rhodomyrtone, a member of the acylphloroglucinols isolated from Rhodomyrtus tomentosa leaves, against important hospital-acquired antibiotic-resistant pathogenic bacteria were assessed. The results indicated that rhodomyrtone exhibited pronounced antibacterial activity against key antibiotic-resistant pathogens including epidemic meticillin-resistant Staphylococcus aureus (EMRSA), vancomycin-intermediate S. aureus and vancomycin-resistant enterococcal strains. The strains EMRSA-16, Enterococcus faecalis ATCC 29212 and VRE-3 demonstrated a significant decrease in survival ability after treatment with rhodomyrtone at 1× (0.5 µg ml(-1)), 2×, 4× and 8× MIC for 24 h. Moreover, the compound was observed in the cytoplasmic fraction of rhodomyrtone-treated S. aureus, and only a very fine band of the compound was seen following separation of the cell-wall and cell-membrane fractions of the treated cells. In addition, exposure of S. aureus to rhodomyrtone at 4×, 2× and 1× MIC for 24 h produced no significant effect on the bacterial cell membrane and cell lysis, suggesting that neither of these is the main target of rhodomyrtone action in these organisms. Stepwise isolation of the bacterial cells with increasing resistance to rhodomyrtone was not induced in either S. aureus or EMRSA-16 after 45 passages on Luria-Bertani agar supplemented with rhodomyrtone. In addition, in vitro toxicity of rhodomyrtone at 128× MIC on human erythrocytes was not observed. These results provide evidence to support therapeutic challenges of rhodomyrtone against Gram-positive pathogens.