The secondary metabolites produced byTricholoma ustaloidesRomagn., a mushroom species belonging to the largeTricholomagenus (Basidiomycota, Tricholomataceae), are unknown. Therefore, encouraged by the interesting results obtained in our previous chemical analyses of a fewTricholomaspecies collected in Italian woods, we aimed to investigate the secondary metabolites ofTricholoma ustaloides. The chemical analysis involved the isolation and characterization of secondary metabolites through an extensive chromatographic study. The structures of isolated metabolites, including the absolute configuration, were established based on a detailed analysis of MS, NMR spectroscopic, optical rotation, and circular dicroism data, and on comparison with those of related compounds reported in the literature. Two novel lanostane triterpenoids, named tricholidic acids B and C, together with triglycerides, a mixture of free fatty acids, five unidentified metabolites, and the known rare saponaceolides F and J, tricholidic acid, and tricholomenyn C, were isolated from an EtOAc extract of fruiting bodies ofTricholoma ustaloidesthat were collected in an Italian beech wood. This is the second example of isolation of tricholidic acid derivatives from a natural source. Saponaceolides F and J exhibited high cytotoxicity (IC50values ≤ 10 μM) against a panel of five human cancer cell lines. The toxicity against myeloid leukemia (HL-60), lung cancer (A-549), hepatocellular cancer (HepG2), renal cancer (Caki-1), and breast cancer (MCF-7) cells was higher than that shown by the very well-known cytotoxic drug cisplatin.