Deoxynivalenol (DON) is a well-known mycotoxin, responsible for outbreaks of gastrointestinal disorders in Japan. Fusarium graminearum, a parasite of cereal crops, produces this toxin and this is one of the reasons why it is important to understand its metabolism. It is possible to predict the mold’s color change and the quantity of DON synthesized throughout its lifecycle. Furthermore, a_w has been found to affect the amount of DON. This study aimed to analyze the potential of F. graminearum surface color as a predictor of DON concentration at a_w = 0.94, 0.97 and 0.99. Thus, 36 specimens were incubated at 25 °C, 12 at each a_w. After 4, 8, 12 and 16 days, 3 specimens from each a_w were collected for color analysis and DON quantification. For color analysis, photos were taken and red, green and blue (RGB) channels were measured on ImageJ software. DON was quantified through liquid chromatography (HPLC). Color changes were only observed at a_w = 0.99 because at lower a_w the molds presented high growth of white mycelium. Yet, DON increased in all cases. It was only possible to relate the colors with DON concentration at a_w = 0.99, where they presented inverse proportionality.