Fusarium graminearum, causal agent of Fusarium head blight (FHB), causes a huge economic loss. No information is available on the activity of quinofumelin, a novel quinoline fungicide, against F. graminearum or other phytopathogens. In this study, we used mycelial growth and spore germination inhibition methods to determine the inhibitory effect of quinofumelin against F. graminearum in vitro. The results indicated that quinofumelin excellently inhibited mycelial growth and spore germination of F. graminearum, with the average EC₅₀ values of 0.019 ± 0.007 μg/mL and 0.087 ± 0.024 μg/mL, respectively. In addition, we found that quinofumelin could significantly decrease deoxynivalenol (DON) production and inhibit the expression of DON-related gene TRI5 in F. graminearum. Furthermore, we found that quinofumelin could disrupt the formation of Fusarium toxisome, a structure for producing DON. Western blot analysis demonstrated that the translation level of TRI1, a marker gene for Fusarium toxisome, was suppressed by quinofumelin. The protective and curative assays indicated that quinofumelin had an excellent control efficiency against F. graminearum on wheat coleoptiles. Taken together, quinofumelin exhibits not only an excellent antifungal activity on mycelial growth and spore germination, but also could inhibit DON biosynthesis in F. graminearum. The findings provide a novel candidate for controlling FHB caused by F. graminearum.