Black spot caused by Alternaria brassicicola is one of the most common fungal diseases of postharvest broccoli. The present study investigated the biocontrol efficacy of Meyerozyma guilliermondii against black spot of postharvest broccoli and the mechanisms involved in the enhanced disease resistance of broccoli based on its effects on reactive oxygen species (ROS) metabolism. Our investigation proved that M. guilliermondii could decrease the disease index of postharvest broccoli caused by A. brassicicola. This antagonistic yeast could potentially increase peroxidase, catalase and superoxide dismutase activities, and the activities of enzymes involved in ascorbate–glutathione cycle and glutathione peroxidase cycle, such as ascorbate peroxidase, glutathione peroxidase and glutathione reductase. Furthermore, the contents of non-enzymatic antioxidants, including ascorbic acid and glutathione, were enhanced by this yeast. Accordingly, the accumulation of ROS, including hydrogen peroxide (H2O2) and superoxide anion (O2–), was reduced by M. guilliermondii, as well as malondialdehyde amount which was one of the membrane lipid peroxides. Our results suggested that M. guilliermondii could improve the activities of ROS scavenging enzymatic system and the levels of antioxidant substances to scavenge excessive ROS, and then protect cells from oxidative damage, which enhanced the disease resistance of broccoli to pathogens. Overall, our study provides a new disease control strategy for postharvest broccoli by improving the ability of ROS scavenging to resist pathogens.