A total methanolic extract of Ginkgo biloba leaves was fractionated by solvent partition using ethyl acetate (fraction A), n-butanol (fraction B) and water (fraction C). The antimicrobial activity of the three fractions was evaluated using a number of Gram-positive and -negative bacteria and yeasts. The apolar fraction A appeared to be the most interesting because of its activity against several microorganisms; this fraction was further separated by high performance liquid chromatography, and shown to contain substances with strong inhibitory activity against Enterococcus faecalis 31, different from the major known chemical components of G. biloba leaves.