BACKGROUNDKiwifruit soft rot is mainly caused by Botryosphaeria dothidea, representing a considerable threat to kiwifruit industry. This investigation assessed the inhibitory consequences and mechanisms of honokiol against B. dothidea, evaluating the inhibitory effects and underlying mechanism.RESULTSA strain of B.dothidea (XFCT‐2) was isolated from infected soft rot kiwifruit. The findings indicate that honokiol hindered the mycelial growth, conidial germination, and pathogenicity of B. dothidea in a dose‐dependent manner, both in vitro and in vivo. Furthermore, ultrastructural examinations showed that honokiol impaired the integrity of B. dothidea, leading to an elevation in cell membrane permeability, engendering a multitude of intracellular substance extravasations and hampering energy metabolism. Transcriptome analysis exhibited that honokiol‐regulated genes were related to membrane lipid biosynthesis, comprising ACC1, FAS2, Arp2, gk, Cesle, and Etnk1. These findings indicate that honokiol impedes B. dothidea by obstructing lipid biosynthesis within the cell membrane and compromising its integrity, halting the growth of the mycelia, which could potentially cause cellular demise.CONCLUSIONThis investigation illustrates how honokiol functions as an eco‐friendly approach to prevent the occurrence of soft rot in kiwifruits. © 2023 Society of Chemical Industry.