Microgreens are a product category with a biochemical content that is currently earning them the status of a functional food. The genotype of the microgreens, and environmental factors, such as the photosynthetic photon flux density (PPFD) and light spectra, can influence the yield and biochemical profile. A landrace of broccoli called ‘Mugnoli’ was compared with a commercial variety (‘Broccolo Natalino’) in two microgreen growing systems (greenhouse vs. growth chamber) and under three growth chamber light spectra (blue, control, control + blue). The results showed that both Mugnoli and Broccolo Natalino can be used to produce microgreens, achieving similar yields, but that Mugnoli showed notably higher polyphenols and antioxidant contents. Due the higher PFFD of the greenhouse environment, microgreens yields were 18% higher than the yields from cultivation in the growth chamber. Regarding the results under different growth chamber spectra, monochromatic blue caused reductions in the microgreens yield and polyphenols content of 13.5% and 14.2%, respectively. In conclusion, Mugnoli can be considered a valuable genetic source for the production of microgreens given its fast crop cycle, good fresh weight production, and, compared to Broccolo Natalino, its superior biochemical content and lower susceptibility to PPFD variations.