This study investigated the impact of Methyl Jasmonate (MeJA) application on the nutritional content and yield of five different colored radish microgreens. Microgreens were produced without substrate and subjected to 0.5 mM and 1.0 mM MeJA treatments on the 7th day, three days before harvest. The parameters measured included yield, dry matter, minerals, amino acids, secondary metabolites such as chlorophylls (Chls), anthocyanins, flavonoids, phenolics, glucosinolates (GSLs), vitamin C, and antioxidant capacity. MeJA at 1.0 mM generally improved yield and dry weight across cultivars, and all microgreens exhibited rich mineral and amino acid composition, with the influence of cultivar being more significant than MeJA treatment. However, MeJA enhanced all cultivars’ anthocyanins, GSLs, phenolics, flavonoids, and antioxidant activities. Generally, as the antioxidant capacity is the primary factor influencing the nutritional quality of microgreens, MeJA-treated microgreens, especially with selected superior cultivars such as ‘Asia purple’ and ‘Koregon red’, could offer a potential for cultivation of value-added, eco-friendly microgreens with substrate-free cultivation.