Thymol is a plant-derived natural compound with antimicrobial activity. However, we have little knowledge about the application of thymol in agriculture. One of the limitations is the high volatility and low aqueous solubility of thymol. Tomato gray mold caused by Botrytis cinerea is one of the most devastating postharvest diseases. In this study, we prepared a nano-emulsion of thymol (named as Nano-Thy) to form a stable O/W (oil in water) microemulsion. In vitro experiments showed that Nano-Thy had antifungal activity against B. cinerea by inhibiting mycelial growth and spore germination. Nano-Thy induced ROS accumulation in mycelia, further leading to lipid peroxidation, cell membrane damage, and subsequent cell death. Nano-Thy significantly prevented the infection of B. cinerea on fresh tomato fruits. Finally, we discussed the mechanisms and their significance in controlling postharvest disease of fruit crops.