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Background: Macrofungi have an established history of use in traditional oriental medicine. Anthracophyllum lateritium is a terrestrial macrofungus found in the dry zone forest reserves in Sri Lanka. Yet there are no scientific reports on bioactive properties of this species. Hence, the current study was aimed at determining the antioxidant potential, in vitro antiproliferative activity and apoptotic effect induced by crude methanolic extract of A. lateritium against RD sarcoma cell line., Method: The crude extract of A. lateritium was dissolved in methanol (MEFCA) and antioxidant activity was evaluated using in vitro assays: inhibition of DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging, ferric ion reducing power and 2-deoxy-D-ribose degradation assay. Total phenol and flavonoid contents of MEFCA were assayed using folin Ciocalteu method and aluminium chloride colorimetric method. In vitro cytotoxicity was determined using MTT assay against RD cells after 24 h exposure to MEFCA. Ethidium bromide/ acridine orange staining, DNA fragmentation and protein synthesis experiments were used to study the apoptotic features and antiproliferative activities of the treated cells. Glutathione assay and griess nitrite assay were used to analyze the reduced glutathione content and liberation of nitric oxide from apoptotic cells., Results: MEFCA showed promising antioxidant activity with EC50 values of 8.00 ± 0.35 μg/mL for DPPH scavenging and 83.33 ± 0.45 μg/mL for 2-deoxy-D-ribose degradation assay. The phenolic content was 265.15 ± 0.46 of (w/w) % of Gallic acid equivalents and flavonoid content was 173.01 ± 0.35 of (w/w) % of Epigallocatechingallate. A. lateritium showed strong in vitro cytotoxic activity with an EC50 of 18.80 ± 4.83 μg/mL for MTT assay against RD cells. Ethidium bromide/acridine orange staining and DNA fragmentation indicated the apoptotic features of treated cells. Protein levels showed a dose dependent decrease supporting the fact that A. lateritium induces apoptosis of treated cells. Glutathione content and nitric oxide content of cells exhibited a dose dependent increase suggesting the apoptosis of RD cells was mediated by both nitrie ions and nitric oxide., Conclusions: The crude extract of the A. lateritium exhibited potent antioxidant, antiproliferative activity and apoptotic effect against RD cells providing supportive evidence for the ethnopharmacological use of this fungus in control of oxidative damage and remedy of cancer.