Microgreens are innovative vegetable products whose production and consumption are gaining popularity globally thanks to their recognized nutraceutical properties. To date, the effects of lighting conditions and growing substrate on the performances of Brassica carinata microgreens (indigenous to Africa) remain underexplored. The present study aimed at providing insights into the influence of different lighting treatments provided by LEDs, namely monochromatic blue (B), red (R), cool white (W) and a combination of three color diodes (B + R + W), and substrates (cocopeat, sand and cocopeat–sand mix (v/v) (1:1)) on the growth, yield and bioactive compounds of B. carinata microgreens. Seeds were germinated in dark chambers and cultivated in growth chambers equipped with LED lighting systems for 14 days under a fixed light intensity of 160 ± 2.5 µmol m⁻² s⁻¹ and photoperiod of 12 h d⁻¹. The best performances were associated with the spectrum that combined B + R + W LEDs and with substrate resulting from the cocopeat–sand mix, including the highest yield (19.19 g plant⁻¹), plant height (9.94 cm), leaf area (68.11 mm²) and canopy cover (55.9%). Enhanced carotenoid and flavonoid contents were obtained with B + R + W LEDs, while the B LED increased the total amount of chlorophyll (11,880 mg kg⁻¹). For plants grown under B + R + W LEDs in cocopeat, high nitrate levels were observed. Our results demonstrate that substrate and light environment interact to influence the growth, yield and concentration of bioactive compounds of B. carinata microgreens.