The use of mushrooms has immense potential in many diverse applications. Until now, more than 3,000 species areconsumed around the world, and more than 100 have shown promising clinical activity against cancer and other chronicdiseases. Sparassis latifolia (formerly S. crispa) is an edible mushroom that harbors β-glucan reported to possessimmunostimulatory and anticancer properties. However there have been no reports on the anticancer activity of hydrophobicfractions of S. latifolia. In this study, the anticancer activities of S. latifolia extract and hydrophobic fractions were investigatedusing AGS (stomach cancer), A529 (lung cancer), and HepG2 (liver cancer) cell lines. In cytotoxicity results of A529 cells, fractionsof A2, A3, A4, A6, A7, A8, A9, and A10 in all 12 fractions show low IC50 values. For HepG2 cells, A7 fraction results in thelowest IC50 value while A7, A8, and A11 fractions show low IC50 values in AGS cells. S. latifolia extract lead to low cell viability incancer cells, compared to positive control of paclitaxel. A compound with molecular weight of 181 were detected using HPLCMSbut not identified yet. As a result, the hydrophobic fractions of S. latifolia EtOH extract would be a possible candidate asnatural anticancer agents in the future.