α-(1→3)-Glucans constitute the least known group of polysaccharides that make up the fungal cell wall. The objective of the study was to assess the biological activity of carboxymethylated derivatives of α-(1→3)-glucans extracted from fruiting bodies of the cultivated species of Pleurotus (P. citrinopileatus, P. djamor, P. erynii, and P. precoce) genus. The polysaccharides extracted from the fruiting bodies were structurally analyzed (using FT-IR and ¹H NMR) and, based on the analysis results, it was proved that they were linear α-(1→3)-glucans. With the use of a carboxymethylation method, the water-insoluble α-(1→3)-glucans were converted into soluble forms, i.e. carboxymethyl-α-(1→3)-glucans (KM-α-(1→3)-glucans). Next, their effect was investigated on the viability of normal CCD 841 CoTr cells (normal human colonic epithelial cells), CCD-18Co (intestinal myofibroblasts), and HeLa tumour cells (human cervical cancer). The metabolic activity, toxicity (MTT and NR), and free radical scavenging ability (DPPH^•) were analyzed. It was shown that the effect of KM-α-(1→3)-glucan derived from the P. citrinopileatus fruiting bodies was the strongest as regards the reduction of the cancer cell viability. Then, the NR method used showed that all the tested KM-α-(1→3)-glucans within the entire concentration range caused the viability of normal cells (CCD 841 CoTr and CCD-18Co) to increase statistically significantly. The KM-α-glucans analyzed did not exhibit any free oxygen radical reduction activity. Based on the results of the differential fluorescent staining analysis, it was confirmed that the KM-α-glucan from fruiting bodies of P. citrinopileatus disturbed the membrane integrity of tumour cells. On the other hand, the fluorescent staining of the F-actin filaments proved that this KM-α-glucan did not have any destructive effect on the cytoskeleton of the cells studied.