Endogenous cytokinins in mycelia of medicinal mushrooms Hericium coralloides and Fomitopsis officinalis grown in vitro were identified using high-performance liquid chromatography coupled with mass spectrometry. High amounts of zeatin-type cytokinins and isopentenyladenine were found. The qualitative composition and quantitative content of cytokinins were species-specific traits of mushrooms. Optical microscopy was used to perform a comparison analysis of the influence of crude extracts and purified cytokinin fractions from both species' mycelial biomass on HepG2 tumor cell growth in vitro and morphology. The results showed that purified cytokinin fractions from H. coralloides and F. officinalis mycelia demonstrated a cytotoxic effect on HepG2 cells, unlike crude extracts. Under the influence of all mushroom extracts, similar patterns of changes in HepG2 cell morphology were observed, but they were more pronounced for H. coralloides compared with F. officinalis. Purified fractions of both mushroom species caused an increased level of apoptosis compared to crude extracts. Some increase in glucose uptake by cultured cells was found in all investigated samples wherein the influence of H. coralloides extracts was approximately twice the effect of the corresponding F. officinalis extracts. The data obtained confirm the assumption that cytokinins are involved in the expression of therapeutic effects of medicinal mushrooms and indicate the need to take into consideration the methods of cytokinin extraction when preparing pharmacologically active drugs based on fungal raw materials. Thus, extracts from H. coralloides and F. officinalis mycelial biomass are promising in the search for anticancer agents.