Kiwifruit is a nutritious and economically important fruit that is widely cultivated in China. In 2021, leaf spot disease of kiwifruit was discovered in the main kiwifruit-producing area of Xifeng County, Guizhou Province, China. Leaf spot disease weakens plant photosynthesis and reduces nutrient synthesis, thereby affecting plant growth. We studied the morphological characteristics and performed a combined analysis of EF-1α, RPB2, and TUB2 genes of Fusarium fujikuroi, a fungus associated with leaf spot disease. The pathogenicity of F. fujikuroi followed Koch’s hypothesis, confirming that this fungus is the cause of kiwifruit leaf spot disease. The sensitivity of seven natural antifungal agents against F. fujikuroi was measured using the mycelial growth rate method. Honokiol, cinnamaldehyde, and osthol showed good antifungal effects against F. fujikuroi, with EC50 values of 18.50, 64.60, and 64.86 μg/mL, respectively. The regression coefficient of cinnamaldehyde was the largest at 2.23, while that of honokiol was the smallest at 0.408. Fusarium fujikuroi was the most sensitive to cinnamaldehyde.