The antimicrobial activity of three propolis ethanol extracts (EEP) was examined for various Gram-negative and Gram-positive bacterial species, including multiple-resistant Staphylococcus aureus, Enterococcus spp. and Pseudomonas aeruginosa strains. EEP had a good bactericidal activity against Gram-positive species, and all multiple-resistant bacterial strains tested were sensitive to EEP. Minimal inhibitory concentrations (MICs) were lower in samples of higher flavonoid content (from 0.65 to 7.81 mg mL(-1)), indicating the influence of the concentration of some potent bactericidal compound(s) in propolis or synergism among some bactericidal compounds. Antimicrobial-guided separation of flavonoid aglycones (bioassay in situ on thin-layer chromatogram) showed that galangin (3,5,7-trihydroxyflavone) is one compound in EEP with bactericidal activity. Galangin was isolated by preparative chromatography. After determining the quantity present, the MIC against multiple-resistant bacteria was determined. The MIC of galangin against multiple-resistant bacterial strains was significantly lower (from 0.16 to 0.44 mg mL(-1), p < 0.05) than that of EEP. The bactericidal activity of galangin against P. aeruginosa strains was present at 0.17+/-0.05 mg mL(-1).