Fomitopsis betulina (Bull.) B.K. Cui, M.L. Han & Y.C. Dai has been used for medicinal purposes for over 5000 years. Numerous studies have confirmed the biological activity of compounds found in this species. The purpose of this study was a comparative analysis of selected groups of metabolites in the extracts from fruiting bodies and mycelial cultures. Phenolic acids (syringic, gallic, p-hydroxybenzoic, 3,4-dihydrophenylacetic), indole compounds (L-tryptophan, 5-hydroxy-L-tryptophan, 5-methyltryptamine), sterols (ergosterol, ergosterol peroxide, hexestrol, cholecalciferol), and triterpenes (betulinic acid, betulin) were determined quantitatively by high performance liquid chromatography with UV-Vis/DAD detection, while fatty acids were assessed with the gas chromatography method. Cytotoxic activity against selected human cancer cell lines was determined using the lactate dehydrogenase test. Anti-inflammatory activity was evaluated on lipopolysaccharide activated A549 cells. Those extracts with anti-inflammatory activity were evaluated for their inhibition of pro-inflammatory enzymes. The mycelium extract exhibited significant cytotoxic activity against prostate cancer cells, while the fruiting body extract indicated a moderate effect on the viability of melanoma and prostate cancer. Incubation of lung epithelial cells with biomass extract significantly decreased cyclooxygenase-2 levels compared to LPS activated A549 cells. This paper is the first report of a comparative quantitative analysis of the metabolites in mycelial cultures and fruiting bodies. In addition, a novel element of this study is its comparison of the cytotoxic and anti-inflammatory activity of the obtained extracts. The results of comparing the composition and activity of mycelium and fruiting bodies shows that the cultures could be proposed as a potential biotechnological source for selected biologically active compounds.