The main purpose of this study was to investigate the effects of three microalgae strains, Chlorella vulgaris, Scenedesmus quadricauda, and Klebsormidium sp. K39, on the enhancement of lettuce yield and soil fertility and in mitigation of nitrogen losses in groundwater, focusing on some aspects of the complex soil-plant system. The experimental trials were carried in pots, and involved the application of microalgae cells at two different concentrations (50 and 500 mg kg− 1 of soil), alone or in combination with standard mineral fertilization. The yield, main morpho-biometric parameters, and protein content of lettuce seedlings, as well as the activities of key enzymes involved in the nitrogen pathway (nitrate reductase, glutamine synthase, and glutamate synthetase) at both root and shoot levels, were monitored and the results were compared to not-inoculated control plants. The nitrate leached due to over irrigation was also evaluated. Furthermore, even the effects of microalgae biostimulants on soil biochemical activity were analysed by monitoring fluorescein diacetate hydrolysis, dehydrogenase, acid and alkaline phosphomonoesterase, and urease activities. All treatments significantly improved lettuce growth, especially when combined with mineral fertilization, providing comparable levels to the control plants treated only with microalgae cells. Furthermore, microalgae treatments positively influenced soil biological activities, as evidenced by increased of the potential biochemical index of soil fertility (Mw). In conclusion, microalgae soil treatments may be considered as a viable strategy to assist growers in reducing the use of mineral fertilizers, with a view to improve plant growth as well as soil biological activity.