Powdery mildew is one of the main problematic diseases in melon production, requiring the use of chemical pesticides with disease-resistant cultivars for control. However, the often rapid acquisition of fungicidal resistance by mildew pathogens makes this practice unsustainable. The identification of crop treatments that can enhance resistance to powdery mildew resistance is therefore important to reduce melon crop attrition. This study indicates that the application of Nano-Se can reduce the powdery mildew disease index by 21–45%. The Nano-Se treatment reduced reactive oxygen species (ROS) and malondialdehyde (MDA) accumulation, with increases in glutathione (GSH), proline and 1,1-Diphenyl-2-picrylhydrazyl radical (DPPH). Increases were also observed in the activities and transcriptional levels of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and peroxidase (POD). Assays with four different cultivars of melon with differing levels of mildew resistance demonstrated that relative to the control, the Nano-Se treatment resulted in larger responses to mildew infection, including increases in the levels of putrescine (PUT; 43–112%) and spermine (SPM; 36–118%), indoleacetic acid (IAA; 43–172%) and salicylic acid (SA; 24–73%), the activities of phenylalanine ammonium lyase (PAL), trans-cinnamate 4-hydroxylase (C4H) and 4-coumarate: Co A ligase (4CL) of the phenylpropanoid pathway (22–38%, 24–126% and 19–64%, respectively). Key genes in the polyamine and phenylpropanoid pathway were also upregulated. These results indicate that the foliar application of Nano-Se improved melon defenses against powdery mildew infection, with a significant reduction in mildew disease development.Graphical