Objective: To explore the therapeutic effects of the novel wild edible mushroom Astraeus hygrometricus (Pers.) Morgan (A. hygrometricus) on human acute lymphoblastic leukemia cells. Methods: Extensive screening of the antiproliferative and chemopreventive potential of different extracts from 5 wild mushrooms, A. hygrometricus, Phallus sp., Lentinus sp., Tricholoma sp., and Serpula sp. was performed against a panel of 6 cancer cell lines and normal cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis determination, cell cycle profiling, intracellular reactive oxygen species (ROS) and reactive nitrogen species (RNS), and mitochondrial membrane potential were analyzed by flow cytometry. The activity of caspases was measured colorimetrically, and the expression pattern of mitochondrial proteins was analyzed. Results: The methanol extract of A. hygrometricus and MOLT-4 cells were identified as the most potent extract exhibiting antiproliferative activity and most sensitive cell line, respectively. The mushroom extract induced robust selective apoptosis in MOLT-4 cells and arrested cell cycle progression at the G0/G1 stage. The extract disrupted the mitochondrial membrane potential and enhanced ROS production in MOLT-4 cells. The methanol extract induced apoptosis by downregulating the expression of Bcl-2, increasing the expression of Bax, and activating the caspase cascade. Conclusion The novel wild edible mushroom is a potential repository of biomolecules for the development of antileukemic drugs.