The inhibitory effects and mechanism of eugenol, carvacrol and thymol against Fusarium graminearum were studied by transcriptomics. The effective concentrations of eugenol, carvacrol and thymol against the mycelium growth, spore germination, and deoxynivalenol biosynthesis of F. graminearum were determined through in vitro antifungal tests. At the cellular level, the action sites were further explored by measuring the conductivity, adenosine triphosphate (ATP) level and malondialdehyde (MDA) level of the mycelium solution treated with each of the three compounds. Thymol had the best antifungal activity among the three compounds. All these compounds could damage the cell membrane integrity of F. graminearum, causing electrolyte leakage and an imbalance in energy metabolism. In addition, they could exert antifungal activity against F. graminearum through regulating the ribosome and mitochondrial structure and enzyme synthesis at the transcriptome level.