Considering the current global climate and demographic conditions, combined with the growing demand for food diversification, the need for innovative functional foods that adhere to the principles of the circular economy is becoming clear. Therefore, this research aims to identify an appropriate cultivation system and growth substrate to maintain a high germination rate and produce basil aromatic microplants with superior quality traits that are entirely edible, together with the substrate. Microplants were grown in both aseptic (AS) and non-aseptic (NAS) systems. Both AS and NAS experiments were conducted in vitro using eco-innovative production technology. Moreover, various growth substrates were tested, such as perlite, agar, banana peel, peat, and their combinations. The analyses focused on the germination capacity, morphometric measurements, and biochemical analyses of the microplants. The results showed that the edible agar-based substrate, used in both AS and NAS, increased the germination capacity up to 95.00 ± 0.30%, while peat provided a germination capacity of only 12.07 ± 1.27% under AS conditions and 6.07 ± 0.35% under NAS conditions. Most biochemical analyses indicated that AS conditions are more suitable for basil microplant production, increasing the dry matter content, total phenolic content, total flavonoid content, and total antioxidant capacity compared to NAS conditions. These findings support the adoption of a new eco-innovative technology that provides organic basil microplants, which are fully usable along with the edible agar substrate.