Mycotoxins, including zearalenone (ZEA), can occur worldwide in cereals. They can enter the food chain and cause several health disorders. ZEA and its derivatives (α-zearalenol, α-ZOL and β-zearalenol, β-ZOL) have structural analogy to estrogen, thus they can bind to estrogen receptors (ERs). In order to characterize the estrogenic activity of ZEA, α-ZOL and β-ZOL, the proliferation of ER-positive human breast cancer cells (MCF-7) exposed to these mycotoxins was measured. After exposure at levels ranging from 6.25 to 25 µM, cell proliferation was evaluated by using the E-Screen bioassay. In accordance with previous studies, our results show the estrogenic activity of ZEA, α-ZOL and β-ZOL in MCF-7 cells. This effect is related to ZEA and its metabolites being flexible enough to bind to mammalian ERs. The relative proliferative effect (RPE) ranged from 10% to 91%. The α-ZOL induced the highest proliferative effect due to its higher affinity for the ERs compared to the other mycotoxins.