Previous studies have reported that 4,6′-Anhydrooxysporidinone (SSF2-2), isolated from Fusarium lateritium SSF2, has neuroprotective effects on the HT-22 hippocampal neuronal cell line. However, the anti-cancer effect of SSF2-2 remains unclear. Here, we examined the viability of MCF-7 human breast cancer cells treated with SSF2-2 or left untreated using a cell viability assay kit. The underlying molecular mechanism was further investigated by Western blotting and immunocytochemistry studies. The results demonstrated that SSF2-2 inhibited the viability of MCF-7 cells. Treatment with SSF2-2 increased the levels of cleaved caspase-9, cleaved caspase-7, poly (ADP-ribose) polymerase (PARP), and LC3B. Additionally, SSF2-2 significantly increased the conversion of LC3-I to LC3II and LC3-positive puncta in MCF-7 cells.