Nano-selenium (Nano-Se), as a biological stimulant, promotes plant growth and development, as well as defense against biotic and abiotic stresses. Glutathione (GSH) is a crucial antioxidant and is also involved in the plant defense response to various stresses. In this study, the efficacy of combined treatment of Nano-Se and GSH (SeG) on the resistance of cucumber plants to Botrytis cinerea was investigated in terms of the plant phenotype, gene expression, and levels of accumulated metabolites using transcriptomic and metabolomic analyses. The exogenous application of SeG significantly enhanced plant growth and increased photosynthetic pigment contents and capacity. Notably, B. cinerea infection was reduced markedly by 41.9% after SeG treatment. At the molecular level, the SeG treatment activated the alpha-linolenic acid metabolic pathway and upregulated the expression of genes responsible for jasmonic acid (JA) synthesis, including LOX (210%), LOX4 (430%), AOS1 (100%), and AOC2 (120%), therefore promoting JA accumulation in cucumber. Intriguingly, the level of cucurbitacin, an important phytoalexin in cucurbitaceous plants, was found to be increased in SeGtreated cucumber plants, as was the expression of cucurbitacin biosynthesis-related genes OSC (107.5%), P450 (440.8%,31.6%), and ACT (414.0%). These genes were also upregulated by JA treatment, suggesting that JA may be an upstream regulator of cucurbitacin biosynthesis. Taken together, this study demonstrated that pretreatment of cucumber plants with SeG could activate the JA signaling pathway and promote cucurbitacin biosynthesis to enhance the resistance of the plants to B. cinerea infection. The findings also indicate that SeG is a promising biostimulant for protecting cucumber plants from B. cinerea infection without growth loss.