ABSTRACT.- Fusarium graminearum sensu stricto (F. graminearum s.s) and Fusarium proliferatum are species frequently isolated from sorghum grains. However, ecophysiology studies of these Fusarium species have not been carried out on sorghum grains. For this reason, the aim of this work was to evaluate the effects of different water activity levels (0.95, 0.98, 0.995) and temperatures (15 ◦C, 25 ◦C, 30 ◦C) on the mycelial growth and mycotoxin production of these species on a sorghum grain substrate. Both species grow under all evaluated conditions, with temperature being the key factor. The optimal growth temperatures were 25-30 ◦C for F. graminearum s.s. and 30 ◦C for F. proliferatum. Each mycotoxin showed a different response to environmental factors. In general, the production of mycotoxins was mainly influenced by aw, with optimal production conditions being more restrictive than growth ones. The highest levels of mycotoxin production (DON, 15-AcDON, 3-AcDON, ZEN and NIV) by F. graminearum s.s were observed at temperatures between 25 and 30 ◦C and aw of 0.98-0.995. Additionally, the results demonstrated that F. proliferatum exhibited narrower temperature and aw ranges for FB1 and FB2 production, with optimal conditions at 30 ◦C and 0.98 aw. The results obtained demonstrate that sorghum grain is susceptible to colonization by these Fusarium species and subsequent contamination with mycotoxins during summer weather conditions. © 2025 Elsevier B.V. All rights are reserved, including those for text and data mining, AI training, and similar technologies.