Pyriofenone demonstrates outstanding efficacy in controlling powdery mildew. We investigated the impact of pyriofenone on the infection processes and cytological features of Blumeria graminis f. sp. tritici on wheat leaves. The preventive application of pyriofenone before inoculation did not inhibit conidial germination but effectively suppressed both appressorial and haustorial formation. Notably, haustorial formation was effectively inhibited, resulting in the complete suppression of successive lesion development and sporulation. Curative application of pyriofenone after inoculation also inhibited lesion expansion and sporulation. Furthermore, it had considerable impact on the morphogenesis of powdery mildew fungus. We observed multi-formed secondary appressoria, shrunken or bifurcated hyphae, abnormal conidiophores, and clubbed conidia-like structures. Subsequently, we employed a histochemical approach to analyze the localization of essential components for the polar growth of fungal hyphae. Pyriofenone induced mislocalization of the actin cytoskeleton, β-glucan and cytoplasmic vesicles, although it did not affect tubulin orientation.