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Anthracnose caused by Colletotrichum musae is a devastating post-harvest disease on bananas, and it leads to serious economic losses. Exploring environmentally friendly natural compounds to manage anthracnose in fruit has been attracting more attention. In the current study, the effect of hinokitiol against C. musae and on banana quality was investigated. Our results showed that hinokitiol exhibited promising antifungal activities both in vitro and in vivo. Importantly, it effectively delayed banana ripening by maintaining weight, color, firmness, and TSS content, and it dramatically reduced the release of ethylene, the respiration rate, and the expression of genes involved in releasing ethylene. Moreover, the activities of defense-related enzymes including SOD, POD, and APX were enhanced by hinokitiol. The mechanism of hinokitiol against C. musae may be attributed to the destruction on the integrity of the plasma membrane, which leads to the leakage of cellular constituents and eventually the cell death of C. musae. Taken together, this study provided substantial evidence that hinokitiol had great potential to be developed as a natural antifungal agent against C. musae, prolonging the storage period of postharvest bananas. • Hinokitiol exhibited promising activity against C. musae both in vitro and in vivo. • Hinokitiol delayed banana ripening and enhanced the antioxidant enzyme activities.s • Hinokitiol destroyed the morphology and the cell membranes integrity of C. musae. [ABSTRACT FROM AUTHOR]