Melatonin acts as a crucial signaling molecule with multiple physiological functions in plant response to abiotic and biotic stresses. However, the impact and regulatory mechanism of melatonin on attenuating tomato fruit fungal decay are unclear. In this study, we investigated the potential roles of melatonin in modulating fruit resistance to Botrytis cinerea and explored related physiological and molecular mechanisms. The results revealed that disease resistance was strongly enhanced by melatonin treatment, and 50 μM was confirmed as the best concentration. Melatonin treatment increased the activities of defense-related enzymes and decreased hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) content with enhanced antioxidant enzyme activities. Moreover, we found that melatonin treatment increased methyl jasmonate (MeJA) content; up-regulated the expressions of SlLoxD, SlAOC, and SlPI II; and reduced the expressions of SlMYC2 and SlJAZ1. We postulated that melatonin played a positive role in tomato fruit resistance to Botrytis cinerea through regulating H<sub>2</sub>O<sub>2</sub> level and JA signaling pathway.