The anti-staphylococcal activity of an ethanol extract of Rhodomyrtus tomentosa and its pure compound, rhodomyrtone, as well as their effects on staphylococcal biofilm formation and biofilm-grown cells were assessed. MIC and minimal bactericidal concentration values of the ethanol extract and rhodomyrtone against planktonic cultures and biofilms of five clinical strains each of Staphylococcus aureus and Staphylococcus epidermidis, and American Type Culture Collection (ATCC) strains of both species, were 32–512 and 0.25–2 μg ml−1, respectively. Results from time–kill studies indicated that rhodomyrtone at a concentration of 4× MIC could reduce the number of Staphylococcus aureus ATCC 25923 and Staphylococcus epidermidis ATCC 35984 cells by 99.9 % within 3 and 13 h, respectively. The ability of rhodomyrtone and the ethanol extract to prevent biofilm formation and kill mature biofilms was assessed: both demonstrated better activity than vancomycin at inhibiting staphylococcal biofilm formation. In addition, the viability of 24 h and 5-day staphylococcal biofilm-grown cells decreased after treatment with the ethanol extract and rhodomyrtone. The ability to reduce biofilm formation and kill mature biofilms occurred in a dose-dependent manner. Scanning electron microscopy clearly confirmed that treatment with rhodomyrtone at 16× MIC could reduce 24 h biofilm formation and the numbers of staphylococci, whilst at 64× MIC this compound destroyed the organisms in the 5-day established biofilm. These results suggest that rhodomyrtone has the potential for further drug development for the treatment of biofilm-forming staphylococcal infections.