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<i>Fusarium graminearum,</i> 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 <i>F. graminearum</i> or other phytopathogens. In this study, we used mycelial growth and spore germination inhibition methods to determine the inhibitory effect of quinofumelin against <i>F. graminearum</i> in vitro. The results indicated that quinofumelin excellently inhibited mycelial growth and spore germination of <i>F. graminearum</i>, 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 <i>TRI5</i> in <i>F. graminearum</i>. 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 <i>F. graminearum</i> 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 <i>F. graminearum</i>. The findings provide a novel candidate for controlling FHB caused by <i>F. graminearum</i>.