Reinforcing and optimizing sustainable food production is an urgent contemporary issue. The depletion of natural mineral resources is a key problem that is addressed by recycling mined potassium and phosphorus, and nitrogen, whose production depends on very high energy input. A closed-loop approach of fertilizer use asserts the necessity for efficient management and practices of organic waste rich in minerals. Human-derived urine is an underutilized yet excellent source for nitrogen fertilizer, and, in this study, processed urine fertilizer was applied to greenhouse soilless cultivation of lettuce (Lactuca sativa L.) cv. Grand Rapids. Biomass increase, biometric parameters, soil plant analysis development (SPAD) index, minerals, and organic acids content of lettuce were analyzed. From eight different urine fertilizer products generated, K-struvite, urine precipitate-CaO, and the liquid electrodialysis (ED) concentrate supported the growth of lettuce similar to that of commercial mineral fertilizer. ED concentrate application led to the accumulation of potassium (+17.2%), calcium (+82.9%), malate (+185.3%), citrate (+114.4%), and isocitrate (+185.7%); K-struvite augmented the accumulation of magnesium (+44.9%); and urine precipitate-CaO induced the highest accumulation of calcium (+100.5%) when compared to the control, which is an added value when supplemented in daily diet. The results underlined the potential of nitrogen- and phosphate-rich human urine as a sustainable source for the fertilization of lettuce in soilless systems.