Aims: To investigate the antimicrobial properties of 6-oxophenolic triterpenoids isolated from Maytenus blepharodes against different micro-organisms and the mode of action on Bacillus subtilis. Methods and Results: The activity of zeylasterone and demethylzeylasterone was evaluated by microdilution method. Zeylasterone showed a higher activity, being active against Gram-positive bacteria (minimum inhibitory concentration 3-20 mu g ml(-1)) and Candida albicans (10 mu g ml(-1)). Killing curves revealed a bacteriostatic effect on B. subtilis that was dependent on the growth phase and inoculum size. Zeylasterone caused cell membrane alterations in B. subtilis, as shown by potassium leakage and formation of mesosome-like structures. However, membrane disruption was not revealed by either LIVE/DEAD Baclight assay or measurement of intracellular constituent efflux. Zeylasterone showed an early effect on N-acetyl-glucosamine and uridine incorporation and later on that of thymidine and leucine. Conclusions: Diverse micro-organisms exhibit sensitivities towards compounds studied. The permeability changes in the cytoplasmic membrane and nonsimultaneous ceasing of macromolecular synthesis suggest that zeylasterone could act on multiple targets on B. subtilis. Significance and Impact of the Study: The activity showed against B. subtilis as a model of spore-forming bacteria would provide valuable information for further studies in the development of 6-oxophenolic triterpenoids as antiseptic and disinfectant properties.