Fomitopsis officinalis is a medicinal mushroom used in traditional European eighteenth and nineteenth century folk medicine. Fruiting bodies of F. officinalis were collected from the natural environment of Świętokrzyskie Province with the consent of the General Director for Environmental Protection in Warsaw. Mycelial cultures were obtained from fragments of F. officinalis fruiting bodies. The taxonomic position of the mushroom mycelium was confirmed using the PCR method. The presence of organic compounds was determined by HPLC-DAD analysis. Bioelements were determined by AF-AAS. The biochemical composition of the tested mushroom material was confirmed with the FTIR method. Antioxidant properties were determined using the DPPH method, and the antiproliferative activity was assessed with the use of the MTT test. The presence of indole compounds (L-tryptophan, 6-methyl-D,L-tryptophan, melatonin, 5-hydroxy-L-tryptophan), phenolic compounds (p-hydroxybenzoic acid, gallic acid, catechin, phenylalanine), and sterols (ergosterol, ergosterol peroxide) as well as trace elements was confirmed in the mycelium and fruiting bodies of F. officinalis. Importantly, a high level of 5-hydroxy-L-tryptophan in in vitro mycelium cultures (517.99 mg/100 g d.w) was recorded for the first time. The tested mushroom extracts also showed antioxidant and antiproliferative effects on the A549 lung cancer cell line, the DU145 prostate cancer cell line, and the A375 melanoma cell line.