Of all the food-contaminating mycotoxins, aflatoxins, and most notably aflatoxin B₁ (AFB₁), are found to be the most toxic and economically costly. Green farming is striving to replace fungicides and develop natural preventive strategies to minimize crop contamination by these toxic fungal metabolites. In this study, we demonstrated that an aqueous extract of the medicinal plant -known as hyssop-completely inhibits aflatoxin production by without reducing fungal growth. The molecular inhibitory mechanism was explored by analyzing the expression of 61 genes, including 27 aflatoxin biosynthesis cluster genes and 34 secondary metabolism regulatory genes. This analysis revealed a three-fold down-regulation of and encoding the two internal cluster co-activators, resulting in a drastic repression of all aflatoxin biosynthesis genes. Hyssop also targeted fifteen regulatory genes, including and , two major global-regulating transcription factors. The effect of this extract is also linked to a transcriptomic variation of several genes required for the response to oxidative stress such as , , , , , , and . In conclusion, hyssop inhibits AFB₁ synthesis at the transcriptomic level. This aqueous extract is a promising natural-based solution to control AFB₁ contamination.