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Sustainable food production has become increasingly important. Soilless cultivation systems offer several advantages, such as water and nutrient use efficiency, and can be implemented where traditional agriculture is impossible. Bioponic systems use locally or regionally available nutrient sources from organic waste streams (either fluid or solid) and can thus contribute to closing nutrient cycles locally. Bioponics harnesses the metabolic processes of microorganisms which release nutrients from organic matter. This study aimed to set up a bioponic system, by using biogas digestate concentrate and biochar as nutrient sources, and promoting nutrient release from the organic sources by including a biofilter in the system. The development of water quality, plant growth, and quality was monitored extensively. In addition, the influence of either the fungal biocontrol agent Trichoderma atrobrunneum or UV-C treatment of the nutrient solution on plant health and growth was investigated. Three cultivation cycles with Lactuca sativa (“HAWKING” Salanova®) in bioponic (BP), hydroponic (HP), and soil (SO) cultivation were performed. The study showed that healthy lettuces could be produced in BP systems, using a biogas digestate concentrate and biochar as nutrient sources, despite salt accumulation in the nutrient solution. In plant sap analyses, lettuces cultivated in BP systems contained less nitrate but more ammonium and chloride. The yield of the lettuces grown in the BP systems was intermediate, compared to the HP and the SO. The fungus, T. atrobrunneum, strain, T720, survived in soil and soilless cultivation systems. Compared to the HP and the SO systems, the shoot height of lettuces grown in the BP system, with the application of Trichoderma, was significantly increased. In SO systems with Trichoderma application, a significantly higher chlorophyll and flavonoid content, but significantly lower shoot height was observed. The fresh weight of lettuce roots was significantly