Abstract The occurrence of fungi and mycotoxins in foods modify sensorial properties and represents a health risk for consumers, and the use of natural antimicrobials may be an alternative to reduce this problem. The objective of this study was evaluate the potential of allyl isothiocyanate (AITC) in inhibit the production of mycotoxins in corn kernels by Aspergillus parasticus, Fusarium tricinctum, Fusarium verticillioides, Alternaria alternata and Gibberela zeae . Kernels were treated with gaseous AITC at 50, 100 or 500 μL/L during 48 h in hermetic flasks. Then, flasks were opened for 24 h and 100 g of corn were inoculated with 10 5 conidia/g of either fungal species. Flasks were kept at 23 °C for 30 d, and corn was processed for mycotoxins quantification (LC-MS/MS) and AITC residual (LC-DAD). Mycotoxins were produced in all control groups (without AITC), while AITC treatments were able to keep the production of 12 mycotoxins at undetectable levels. AITC penetrated the kernels, and residual doses (∼16%) were detected after 30 d, showing an extended protection period. Gaseous AITC could potentially be used as a prophylactic agent to avoid mycotoxin production in corn.