The culture filtrate of Lentinula edodes showed potent antimicrobial activity against the plant pathogenic bacteria and fungi. To purify antibacterial compound, the culture filtrate was subjected to Octadecyl-silica (ODS) column chromatography and was eluted with different concentrations from 5 to 100% methanol and each 50 μL aliquot of the methanol elutes did not show antimicrobial effect on the bacteria, but the water fraction that passed through the column maintained antibacterial activity on the bacteria. The pH of the passed fraction was 3.9, speculating the low pH can be caused by an organic acid. Qualitative analyses of organic acids from culture filtrate of L. edodes were carried out by high-performance liquid chromatography(HPLC). Nine organic acids including oxalic acid, phytic acid, malonic acid, and fumaric acid were identified and oxalic acid was a dominant component in culture filtrate of L. edodes and it made up 50% from HPLC analysis data. Oxalic acid concentrations over 300mg/L exhibited antimicrobial activities on eight phytopathogenic bacteria and different fungi. The quantitative analysis by HPLC revealed that the content of oxalic acid was more highly detected in water extract from spent mushroom substrate than that of liquid culture.