Mango anthracnose, mainly caused by Colletotrichum gloeosporioides, is the major destructive postharvest disease of mango during storage and transport. Dimethyl trisulfide (DMTS), an organic volatile found in some microorganisms or plants, inhibited growth of C. gloeosporioides in vitro, but its effects on mango anthracnose and its molecular mechanisms of action have not been well characterized. In this study, the EC50 of DMTS against Colletotrichum spp. from mango mainly ranged from 2.3 to 20.0 μL/L. In vivo, the fumigation rates of 20 μL/L of DMTS for 24 h, or 80 μL/L for 3 h or 6 h could effectively reduce severity of anthracnose (natural inoculum) on postharvest mangoes with inhibitory effects of 61.7 %, 65.7 %, and 69.4 %, respectively, as observed 10 days after treatment. Furthermore, there was no detectable DMTS residue in mango skin or flesh, and an overall improvement in the quality of the fruit with higher soluble solids, total sugars, vitamin c, and β-carotene, and lower titratable acidity than the non-treated control. In addition, DMTS could significantly reduce ergosterol content in mycelia of C. gloeosporioides, and gene expression analysis showed DMTS significantly suppressed expression of ergosterol biosynthesis-related genes Cgerg6 and Cgerg11 after mycelia were exposed to DMTS. Knock-out mutants for each of these two genes showed reduced sensitivity to DMTS. After gene complementation in situ, the sensitivity of complementary transformants to DMTS was restored to that of the parental strain. Therefore, we concluded that the genes Cgerg6 and Cgerg11 are involved in an interaction with the antifungal activity of DMTS. This is the first study to demonstrate a control effect of DMTS on mango postharvest anthracnose resulting in reduced disease severity and enhanced fruit quality. Transformant studies also revealed some potential molecular mechanisms of the antifungal activity of DMTS that may lead to improved management of mango postharvest anthracnose.