Hydroponics is a contemporary agricultural system providing precise control over growing conditions, potentially enhancing productivity. Biofertilizers are environmentally friendly, next-generation fertilizers that augment product yield and quality in hydroponic cultivation. In this study, we investigated the effect of three bio-fertilizers in a hydroponic floating system, microalgae, plant growth-promoting rhizobacteria (PGPR) and arbuscular mycorrhizal fungi (AMF), combined with a 50% reduction in mineral fertilizer, on lettuce yield and quality parameters including antioxidants: vitamin C, total phenols and flavonoids. The treatments tested were: 100% mineral fertilizer (control 1), 50% mineral fertilizer (control 2), 50% mineral fertilizer with microalgae, 50% mineral fertilizer with PGPR and 50% mineral fertilizer with AMF. The research was conducted during the winter months within a controlled environment of a glasshouse in a Mediterranean climate. The PGPR comprised three distinct bacterial strains, while the AMF comprised nine different mycorrhizal species. The microalgae consisted of only a single species, Chlorella vulgaris. AMF inoculation occurred once during seed sowing, while the introduction of PGPR and microalgae occurred at 10-day intervals into the root medium. Our findings revealed that the treatment with PGPR resulted in the highest growth parameters, including the lettuce circumference, stem diameter and fresh leaf weight. The 100% mineral fertilizer and PGPR treatments also yielded the highest lettuce production. Meanwhile, the treatment with AMF showed the highest total phenol and flavonoid content, which was statistically similar to that of the PGPR treatment. Furthermore, the PGPR recorded the maximum range of essential nutrients, including nitrogen (N), potassium (K), iron (Fe), zinc (Zn) and copper (Cu). Thus, the inclusion of PGPR holds promise for optimizing the lettuce growth and nutrient content in hydroponic systems. In conclusion, PGPR has the potential to enhance nutrient availability in a floating hydroponic system, reducing the dependence on chemical fertilizers. This mitigates environmental pollution and fosters sustainable agriculture.