Abstract: The key factors in the antifungal activity of Hypocrellin A (HA), the metabolites of Shiraia bambuscola, against the Botrytis cinerea were investigated. The type of photoactivity of HA was determined based on the principle of active oxygen quenching. The correlation between the concentration of three reactive oxygen species, including singlet oxygen (¹O₂), hydroxyl radical (·OH), and the superoxide anion radical (\rm O_2\bar\cdot), and the inhibition rate of HA against B. cinerea was analyzed by the singlet oxygen capture principle, Fenton reaction, and riboflavin photochemical reaction. Results showed that the antifungal activity of HA against B. cinerea resulted from the active oxygen generated by photodynamic action (Type I and Type II). Both types exhibited controlling effects against the growth of B. cinerea, but Type II exhibited is more active. The concentration of ¹O₂, ·OH, and\rm O_2\bar\cdotare positively correlated with the bacteriostatic rates, and those three active oxygen species were the key factors in the antifungal activity. Stepwise regression analysis revealed that ¹O₂ is the most critical antifungal factor produced by HA, followed by\rm O_2\bar\cdotand ·OH. The optimal regression model was obtained as follows: Y = 0.055 9 + 0.814 5X₁ + 0.002X₂ + 0.738 4X₃, in which Y was the inhibition rate, X₁, X₂ and X₃ were the concentration of ¹O₂, ·OH and\rm O_2\bar\cdot, respectively, R² = 0.961 9. In this paper, the key factors in the antifungal activity of HA against B. cinerea were investigated for the first time, which laid a foundation for the study of the mechanism of HA and provided a theoretical basis for the development and utilization of HA as a photoactivated pesticide.