One Helicosporium strain, isolated from a wilted chestnut tree, evidenced in vitro antimicrobial activity against various types of bacteria and fungi, and generated a diffusible pigment. The antimicrobial compounds and the diffusible pigment of the Helicosporium sp. isolate were purified via solvent fractionation, column chromatography, and recycling preparative chromatography. Both the major antimicrobial compound and the diffusible pigment were identified as 2-methylresorcinol via nuclear magnetic resonance spectroscopy. Therefore, 2-methylresorcinol, a diffusible pigment generated by Helicosporium sp., appears to be an active antimicrobial principle. This pigment also exhibited considerable cytotoxicity against mammalian cells.