Licochalcone A was isolated from the roots of licorice, Glycyrrhiza inflata, which has various uses in the food and pharmaceutical industries; isolation was followed by extraction with ethanol and column chromatography with silica gel. In this study, the activities of licochalcone A against some food contaminant microorganisms were evaluated in vitro. The vegetative cell growth of Bacillus subtilis was inhibited in a licochalcone A concentration-dependent manner and was completely prevented by 3 μg of licochalcone A/ml. Licochalcone A showed a high level of resistance to heating at 80 to 121°C for 15 min. Licochalcone A did not inhibit the germination of heat-treated spores of B. subtilis induced by l-alanine. Licochalcone A showed effects against all gram-positive bacteria tested and especially was effective against all Bacillus spp. tested, with MICs of 2 to 3 μg/ml, but was not effective against gram-negative bacteria or eukaryotes at 50 μg/ml. Although the cationic antimicrobial peptides protamine and ε-poly-l-lysine resulted in the loss of antimicrobial activity in the presence of either 3% (wt/vol) NaCl or protease at 20 μg/ml, the antibacterial activity of licochalcone A was resistant to these conditions. Thus, licochalcone A could be a useful compound for the development of antibacterial agents for the preservation of foods containing high concentrations of salts and proteases, in which cationic peptides might be less effective.