Sustainable agriculture is currently trendy. It is supported not only for the urban environment but also as an innovation of conventional practices in order to increase the efficiency and quality of agricultural production. This study presents the results achieved within selected soil-less (hydroponic and aeroponic) systems. Then, it compares them, using the tool of comparative life cycle assessment (LCA), with the results of soil cultivation. The attention is directed towards biomass production and the content of bioactive substances, which can compensate for higher operating costs of soil-less cultivation systems. Coffea arabica has shown a significant increase of caffeine and theobromine contents, both in leaves and roots, as well as higher biomass yield during the aeroponic cultivation. On the contrary, Senecio bicolor evinced the results of a considerably increased growth in the hydroponic system, with no higher contents of alkaloid or flavonoids, except for the rutin concentration. The LCA results of the compared soil and soil-less systems showed that the consumption of fertilizers, diesel, and water in soil systems and of conventional electricity in aeroponics and hydroponics contributed mostly to their environmental burden. The major environmental impact categories are terrestrial ecotoxicity, human non-carcinogenic toxicity, and global warming. Therefore, in order to make the soil-less cultivation systems sustainable, these environmental aspects need to be considered deeply.