To develop environment-friendly agricultural products with anti-microbial activity against Sclerotinia sclerotiorum as a pathogen of sclerotium disease, Usnea longissima was extracted by methanol and its extract was fractionated into several solvent fractions. The chloroform fraction, which showed the highest antimicrobial activity, was separated by silica gel-column chromatography and obtained into nine group subfractions. The nine group fractions were searched the antifungal activities by bioassay. The most active No. 3 subfraction was analyzed by GC-MS. Each mass spectra, corresponding to each peak of chromatogram, was compared to database of Wiley library. As a result, Usnic acid was identified as main compounds. In conclusion, Usnic acid isolated from Usnea longissima was antimicrobial chemical against Sclerotinia sclerotiorum as a pathogen of sclerotium disease.