Currently, chemical agents hold great promise in preventing and combating Botrytis cinerea. However, the antifungal mechanism of some agents for B. cinerea remains rather vague, imposing restrictions on the research and development of novel antifungal inhibitors. In this work, we discovered that mulberrin (MBN), a natural compound from the root bark of Ramulus Mori, with an IC50 of 1.38 μM together, demonstrated marked anti-14α-demethylase (CYP51) activity through high throughput virtual screening and in vitro bioactivity assay. The computational biology results demonstrated that MBN and its derivatives were bound to the catalytic activity region of CYP51, but only MBN could form a strong π-cation interaction with the Fe ion of heme in CYP51 via the 2-methylpent-2-ene moiety at atom C9. MBN had a stronger binding free energy than the other three compounds with CYP51, implying that the 2-methylpent-2-ene moiety at atom C9 is a critical pharmacophore for CYP51 inhibitors. Subsequently, through an antifungal test, MBN demonstrated excellent anti-B. cinerea activity by inhibiting CYP51 activity. The EC50 values of MBN toward hyphal growth and spore germination in B. cinerea were 17.27 and 9.56 μg mL-1, respectively. The bioactivity loss of CYP51 by direct interaction with MBN induced the increase of cell membrane permeability, membrane destruction, and cell death. Meanwhile, in the B. cinerea infection model, MBN significantly prolonged the preservation of strawberries by preventing B. cinerea from infecting strawberries and could be used as a potential natural preserving agent for storing fruits.