The aim of this study was to evaluate the effects of two variants of the Daedaleopsis confragosa fungus extract, F-1368 strain, differing in polysaccharide concentration, on five human cancer cell lines (U-87 MG, C-33 A, SK-Mel-28, MDA-MB-231, SW620) in vitro. Standard cytotoxicity assessment methods, including MTT-assay and clonogenic assay, were employed. Additionally, an experiment was conducted on laboratory SCID mice with heterotopic xenografts of human glioblastoma U-87 MG, where the test preparations were administered subcutaneously into the tumor area. Tumor sizes were measured using a caliper, and upon euthanasia, xenografts were histologically examined with hematoxylin-eosin staining under a light microscope. Results from MTT and clonogenic assays demonstrated that F-1368 extracts reduced the viability, mitochondrial function, and proliferative activity of tumor cells in vitro. However, a threefold increase in polysaccharide concentration in one of the extracts did not significantly enhance its cytotoxicity against tumor cells in vitro. Furthermore, one extract was tested on U-87 MG cell xenografts, revealing a reduction in tumor growth in SCID mice. The maximum tumor growth inhibition index for U-87 MG cells reached 50.7% at 21 days post-commencement of extract injections. These findings suggest that the D. confragosa F-1368 strain holds promise for investigating both in vitro and in vivo models of antitumor activity and identifying potential bioactive molecules or compounds.