Fusarium verticillioides is a plant pathogenic fungus that can cause disease in a variety of crops, including corn, sorghum, and cotton. It is commonly found in soil, but can also be found in infected seeds and plant debris. This fungus produces several mycotoxins, including fumonisins, which can be harmful to both humans and animals if ingested in high concentrations. It can also produce other toxins such as "emerging" mycotoxins like beauvericin, fusaproliferin and others. In this study, we aimed to expand our understanding of mycotoxin production by F. verticillioides according to the substrate. To do so, we investigated the ecophysiology of F. verticillioides under different culture conditions (media, humidity, temperature) over time through UHPLC-HRMS/MS analysis and a dereplicative approach. Our results showed a modification of the fungal growth but also a variation of the nature and the relative concentrations of the mycotoxins produced along the time according to the modifications of culture conditions. This experiment showed that corn meal agar (CMA) medium (MEA) was favorable for fumonisin A1 (FA1), fumonisin B1 (FB1) and fumonisin B2 (FB2) production while malt extract agar medium was favorable for fumonisin A2 (FA2) production. This study allowed us to understand the development conditions of this fungus and the occurrence of its mycotoxins depending on its substrate.