In this study, antioxidant and antimicrobial activities of ethanolic crude extract and its five different solvent subfractions (namely, ethyl acetate fraction, n-butanol fraction, chloroform fraction, n-hexane fraction and the aqueous fraction) from a mixture of four different medicinal herbs (Galla rhois, Achyranthes japonica Nakai, Terminalia chebula Retz and Glycyrrhiza uralensis) were investigated. Among all the tested mixture combination of four medicinal herbs, 5:3:1:1 ratio of Galla:Achyranthes : Terminalia : Glycyrrhiza had the best antimicrobial effects against four strains of microorganisms (Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa and Escherichia coli) and exhibited the highest DPPH radical-scavenging activity. Further sub-fractions with solvents were screened for antioxidant and antimicrobial activities. Antioxidant activity in order was ethyl acetate fraction > n-butanol fraction > chloroform fraction > nhexane fraction > aqueous fraction. The n-butanol extracted fraction showed the highest level of antimicrobial activity in com- parison to other fractions. In addition, all those fractions did not show any cytotoxicity against human skin cell CCD-986sk. These results suggest that 5:3:1:1 combination extracts of medicinal herbs (Galla : Achyranthes : Terminalia : Glycyrrhiza) may be potentially used as a safe natural antimicrobial preservative.