Piptoporus betulinus is a fungus known for its medicinal properties. It possesses antimicrobial, anti-inflammatory, and anti-cancer activity. In this study, several tests were performed to evaluate the cytotoxic effect of the ethanolic extract of Piptoporus betulinus on two melanoma human cell lines, WM115 primary and A375 metastatic cell lines, as well as Hs27 human skin fibroblasts. The extract proved to affect cancer cells in a dose-dependent manner, and at the same time showed a low cytotoxicity towards the normal cells. The total phenolic content (TPC) was determined spectrophotometrically by the Folin-Ciocalteu method (F-C), and the potential antioxidant activity was measured by ferric-reducing antioxidant power (FRAP) assay. One of the active compounds in the extract is betulin. It was isolated and then its cytotoxic activity was compared to the results obtained from the Piptoporus betulinus extract. To further understand the mechanism of action of the extract’s anticancer activity, tests on model cell membranes were conducted. A model membrane of a melanoma cell was designed and consisted of 1,2-dimyristoyl-sn-glycero-3-phosphocholine, disialoganglioside-GD_1a and cholesterol: DMPC:GD_1a:chol (5:2:3 mole ratio). Changes in a Langmuir monolayer were observed and described based on Π-A_mol isotherm and compressibility modulus changes. LB lipid bilayers were deposited on a hydrophilic gold substrate and analyzed by IR and X-ray photoelectron spectroscopy. Our study provides new data on the effect of Piptoporus betulinus extract on melanoma cells and its impact on the model of melanoma plasma membranes.