Purification and study of the antibiotic substances produced by Lentinus squarrosulus have been carried out. The substances, excreted in the culture medium, were extracted with n-butanol. The butanolic extract inhibited growth of Rigidoporus lignosus, the agent of white rot of Hevea brasiliensis, and also of Mucor ramannianus, Rhodotorula mucilaginosa, Saccharomyces cerevisiae, and Bacillus subtilis. The antibiotic fractions were purified by silicic acid column chromatography, then by reversed-phase (C18) high performance liquid chromatography (HPLC) followed by preparative adsorption thin-layer chromatography on silica gel. Two purified compounds were obtained: Ls1, which was active against B. subtilis, and Ls2, which in addition was also active against R. lignosus, M. ramannianus, and yeasts. Only the Ls2 compound is analyzed in this work. It was characterized by chemical reactions, ultraviolet spectroscopy, infrared spectroscopy, and proton nuclear magnetic resonance spectroscopy, which indicated the hydrophilic character of the molecule and the presence of alcoholic functions as well as a glycosidic moiety. The properties differed from those of already known antibiotics produced by different species of Lentinus.