Quality formation and disease resistance are important agronomic traits for fruit crops, thereby exhibiting both scientific significance and application values for in-depth studies. However, practical measures simultaneously modulating fruit ripening and disease resistance are seldom reported now. In the present study, it was found that 12-hydroxydodecanoic acid (12-HA), a mid-chain fatty acid, delayed fruit ripening by suppressing ethylene biosynthesis and signaling, while the activities of wall degrading enzymes and the expression levels of related genes decreased upon 12-HA treatment. Treatment with 12-HA did not significantly affect titratable acid, total soluble solids and vitamin C content of tomato fruit. Meanwhile, 12-HA displayed in vitro antimicrobial activities and attenuated pathogenicity of Botrytis cinerea on harvested fruit in a dose-dependent manner. Collectively, 12-HA delayed fruit ripening and inhibited pathogenicity of fungal pathogens on harvested fruit, which may be promising for postharvest handling of fresh produces in agricultural and food industry.