The aims of this study were to evaluate the antifungal activity of the bioactive compound allyl isothiocyanate (AITC) against Aspergillus flavus (8111 ISPA) aflatoxins (AFs) producer and Penicillium verrucosum (D-01847 VTT) ochratoxin A (OTA) producer on corn, barley, and wheat. The experiments were carried out initially in a simulated silo system for laboratory scale composed of glass jars (1 L). Barley and wheat were contaminated with P. verrucosum and corn with A. flavus. The cereals were treated with a hydroxyethylcellulose gel disk to which 500 µL/L of AITC were added; the silo system was closed and incubated for 30 days at 21 °C. After that, simulated silos of 100 L capacity were used. Barley, wheat, and corn were contaminated under the same conditions as the previous trial and treated with disks with 5 mL of AITC, closed and incubated for 90 days at 21 °C. In both cases, the control test did not receive any antifungal treatment. The growth of the inoculated fungi and the reduction in the formation of AFs and OTA were determined. In the lab scale silo system, complete inhibition of fungal growth at 30 days has been observed. In corn, the reduction of aflatoxin B1 (AFB1) was 98.5%. In the 100 L plastic drums, a significant reduction in the growth of A. flavus was observed, as well as the OTA formation in wheat (99.5%) and barley (92.0%).