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