Aims As a natural antimicrobial agent, Melaleuca alternifolia oil (MAO) is generally recognized to be safe and effective in the inhibition of phytopathogenic fungi. Due to lack of comprehensive studies on MAO for controlling postharvest Aspergillus, we investigated the preservative mechanism of MAO and its components against Aspergillus ochraceus in postharvest grapes to evaluate their potential effectiveness as fruit preservatives. Methods and Results In our study, the compositions in MAO were analysed by gas chromatography-mass spectrometry. The inhibitory effects of MAO and its main constituents against A. ochraceus were compared by scanning electron microscopy and transmission electron microscopy observation, and metabolic analysis. Two components of MAO, alpha-terpineol and terpene-4-alcohol, showed higher antifungal effects than MAO, of which alpha-terpineol caused the worst leakage of cytoplasm and most serious hyphae distortions and spore disruptions. The downregulation of metabolic pathways of A. ochraceus was strongest with alpha-terpineol. The best inhibitory efficacy against A. ochraceus in grapes also occurred with alpha-terpineol. 3-Carene showed little inhibitory effect. Conclusions These results demonstrate that not all components in MAO possess antimicrobial effects, and alpha-terpineol is the main contributor of MAO's A. ochraceus inhibition effect. Significance and Impact of the Study alpha-Terpineol may be used as an alternative natural preservative for the postharvest storage of grapes and other fruits.