This study aimed to reveal the effects and possible mechanism of terpinen-4-ol, the main component of tea tree oil (TTO), on the disease resistance of strawberry fruit. When comparing effects of TTO and its components on decay development in fruit inoculated with Botrytis cinerea after treatment, strawberry treated with terpinen-4-ol showed the lowest disease incidence (44.4%) after 48 hours, and also smallest lesion diameter during the whole storage. This indicates that terpinen-4-ol induces the highest disease resistance in strawberry compared with TTO and other components. Untargeted metabolomic analysis showed that terpinen-4-ol treatment strongly activated phenylpropanoid biosynthesis and flavonoid metabolism pathway by increasing the accumulation of cinnamaldehyde, coniferyl aldehyde, naringenin, taxifolin, quercetin and quercitrin in fruit at 12 h after treatment. In addition, terpinen-4-ol treatment also caused the accumulation of total phenolics and lignin by enhancing activities and relative gene expression of key enzymes in the phenylpropanoid metabolism pathway. These results suggest that terpinen-4-ol, as the key component of TTO, is the most important contributor to the effectiveness of TTO in improving disease resistance of strawberry fruit through activating the phenylpropanoid metabolism pathway.