Agronomic factors can affect mycotoxin contamination of maize, one of the most produced cereals. Maize is usually harvested at 18% moisture, but it is not microbiologically stable until it reaches 14% moisture at the drying plants. We studied how three agronomic factors (crop diversification, tillage system and nitrogen fertilization rate) can affect fungal and mycotoxin contamination (deoxynivalenol and fumonisins B1 and B2) in maize at harvest. In addition, changes in maize during a simulated harvest-till-drying period were studied. DON content at harvest was higher for maize under intensive tillage than using direct drilling (2695 and 474 μg kg−1, respectively). We found two reasons for this: (i) soil crusting in intensive tillage plots caused the formation of pools of water that created high air humidity conditions, favouring the development of DON-producing moulds; (ii) the population of Lumbricus terrestris, an earthworm that would indirectly minimize fungal infection and mycotoxin production on maize kernels, is reduced in intensive tillage plots. Therefore, direct drilling is a better approach than intensive tillage for both preventing DON contamination and preserving soil quality. Concerning the simulated harvest-tilldrying period, DON significantly increased between storage days 0 and 5. Water activity dropped on the 4th day, below the threshold for DON production (around 0.91). From our perspective, this study constitutes a step forward towards understanding the relationships between agronomic factors and mycotoxin contamination in maize, and towards improving food safety.