The mycotoxin alternariol (AOH) can be found in food products infected byAlternariaspp. and is considered an endocrine-disruptive mycotoxin. The main mechanism of AOH toxicity is associated with DNA damage and modulation of the inflammation process. Still, AOH is considered as one of the emerging mycotoxins. In this study, we have evaluated how AOH might affect the local steroidogenesis process in the prostate, in both normal and cancer cells. We have found that AOH itself modulates the cell cycle, inflammation, and apoptosis, rather than the steroidogenesis process in prostate cancer cells; however, in the presence of another steroidogenic agent, the influence on steroidogenesis is significant. Therefore, this is the first study to report the effect of AOH on local steroidogenesis in normal and prostate cancer cells. We postulate that AOH might modulate the release of the steroid hormones and expression of the key components by interfering with the steroidogenic pathway and might be considered a steroidogenesis-altering agent.