Apple Valsa canker is one of the most serious diseases, having caused significant apple yield and economic loss in China. However, there is still no effective biological methods for controlling this disease. Our present study focused on the inhibitory activity and mechanisms of Trichoderma longibrachiatum (T6) fermentation on Valsa mali that causes apple Valsa canker (AVC). Our results showed that the T6 fermentation exhibited effective antifungal activity on the mycelial growth and conidia germination of V. mali, causing mycelium malformation and the hyphal disintegrating in comparison to the control. The activity of pathogenically related enzymes that are secreted from V. mali and the expression level of gene of V. mali were significantly inhibited and downregulated by treatment with T6 fermentation. In addition, the lesion area and number of pycnidia of V. mali formed on the branches were significantly reduced after treatment with the T6 fermentation through the pathogenicity test on the detached branches. Our results indicate that the possible mechanism of T6 fermentation against V. mali occurs through inhibiting its growth and reproduction, the pathogenic enzyme activity, and its related gene expression.