In modern agricultural production, cattle manure waste recovery is considered as a sustainable approach to agricultural waste management, reducing environmental pollution and chemical fertilizer use. This study aimed to investigate the effects of manure and digestate derived from a pilot-scale livestock waste-recycling system, in combination with a low copper concentration as a fungicide, on the physiological response of lettuce cv Rufus (Lactuca sativa L.) plants and the associated soil microbiome. A five-week microcosm experiment was conducted in a greenhouse under environmental conditions. Lettuce plant performance was assessed in terms of biomass, leaf area index, photosynthetic activity, chlorophyll measurements, lipid peroxidation, total phenolic content, and nutrient uptake. The results suggested that incorporating digestate into the potting soil mix significantly enhanced crop yields compared to the control and manure treatments. The soil microbial activity increased in the presence of fertilizers, improving the soil chemical and biological properties. The addition of copper negatively affected the growth and physiological performance of the lettuce plants under both the control and manure-treated conditions, except for those grown in the presence of digestate, where copper accumulation was reduced. These findings highlight the potential of growing horticultural crops using organic fertilization through livestock waste anaerobic digestate, establishing a waste-to-food recycling system.