Piptoporus betulinus (Bull.: Fr.) P. Karst. (Fomitopsidaceae) has been commonly used in folk medicine as an antiparasitic and antimicrobial agent in the treatment of wounds and for the treatment of rectal cancer and stomach diseases. Tea obtained from this mushroom has antibacterial, antifatiguing, immunoenhancing, and soothing properties. The purpose of the present study was the evaluation of in vitro anticancer activity of fraction isolated from P. betulinus. The effect on cell proliferation, motility, and viability was assessed in a range of cancer and normal cells. P. betulinus fraction prepared from dried fruiting bodies was subjected to anticancer evaluation in human lung carcinoma (A549), colon adenocarcinoma (HT-29), and rat glioma (C6) cell cultures. Human skin fibroblasts (HSF), bovine aorta endothelial cells (BAEC), models of rat oligodenrocytes (OLN-93), hepatocytes (Fao), rat astroglia, and mouse neurons (P19) were applied to test toxicity in normal cells. The following methods were applied: tumor cell proliferation (MTT assay and BrdU assay), cytotoxicity (LDH assay), tumor cell motility (wound assay), tumor cell morphology (May-Grünwald-Giemsa staining), and death detection (ELISA). P. betulinus fraction elicited anticancer effects that were attributed to decreased tumor cell proliferation, motility, and the induction of morphological changes. Of note is the fact that it produced no or low toxicity in tested normal cells.