Bioactive complexes of medicinal mushrooms have become attractive as complementary anticancer remedies. Our in vitro study focused on the cytotoxicity of the polyphenol-reach and beta-glucan-containing aqueous alkali extract from Fomes fomentarius fruiting bodies (FFE) using murine fibroblasts (L929), human colon adenocarcinoma cells (Caco-2), and cutaneous melanoma cells (COLO-818). Dose-dependent FFE cytotoxicity with an half maximal inhibitory concentration of 0.44 mg/mL was observed for L929 cells upon analysis of the total number of adherent cells, degree of cell viability, cell morphology, and mitochondrial metabolic activity. Cytotoxic effects on cancer cells tested using cell impedance were dependent on FFE concentration, type of cells, and their density. As a routine in vitro model for predicting human intestinal absorption, Caco-2 cells did not react on FFE, which can indirectly support its safety for the human intestinal epithelium. Melanoma cells were affected in a dose-dependent manner, even at low FFE concentrations (0.01-0.05 mg/mL). The confluent cell layer, which resembles a fully formed tumor, was much more resistant than the incompletely formed, subconfluent cell layer, simulating tumor formation. FFE applied topically could be a promising candidate to prevent melanoma development in its early stages.