Several studies have suggested a potential antitumor effect of 2-dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione (DMDD). To further understand the mechanism of action of this compound, we investigated its effect on the phosphatidylinositol-3-kinase (PI3K)/serine-threonine kinase (Akt)/mammalian target of rapamycin (mTOR) signaling pathway. We show that DMDD application significantly inhibited the proliferation of breast cancer cell lines MDA-MB-231 and ER-α positive MCF-7. Furthermore, DMDD application resulted in increased intracellular reactive oxygen species (ROS) levels, apoptosis and autophagy, whereas it downregulated the expression of PI3K, Akt and mTOR mRNA and proteins, and increased the expression of LC3II/I and p62 proteins. In a mouse breast cancer xenograft model, DMDD inhibited tumor growth. Expression analyses suggest that ROS levels were higher in DMDD treated tumor tissues, whereas immunohistochemical analyses suggest that apoptotic cells were more prevalent in the DMDD treated group compared to the control group. Taken together, our results suggest that the molecular mechanism of action of DMDD may involve the enhancement of breast cancer autophagy through the PI3K/Akt/mTOR signaling pathway by mediating ROS expression.