Purpose of work To show biological activity of carboxymethylated α-(1 → 3)-d-glucans isolated from the selected macromycetes fungi on human tumor and normal cells. Water-insoluble, alkali-soluble polysaccharides (WIP) were isolated from fruiting bodies of four macromycetes fungi: Lentinus edodes, Pleurotus ostreatus, Piptoporus betulinus and Laetiporus sulphureus. The structure of the polysaccharides was determined using composition analysis, methylation analysis, fourier transform infrared spectroscopy, and nuclear magnetic resonance spectroscopy. The chemical and spectroscopic investigations indicated that the polysaccharides were an α-(1 → 3)-d-glucans. A biological activity analysis of the carboxymethylated (CM) α-(1 → 3)-d-glucans was based on an assessment of their cytotoxic, mitochondrial metabolism-modulating, and free radical scavenging effects. The cytotoxic activity of the CM-glucans was concentration- and cell-type-dependent. The tested CM-glucans, generally, did not have a free radical scavenging effect. The CM-α-(1 → 3)-d-glucans isolated from the selected macromycetes fungi are biologically active and may therefore be used as diet or therapy supplements.