In the last decades, the number of studies about the valorization of agricultural by-products has strongly increased due to the growing focus on the circular economy. In this context, mushroom cultivation is raising attention for its nutritional properties and adaptability to different growing conditions. The goal of this project is, therefore, to create a link between two production chains, valorizing the solid anaerobic digestate fraction from the biogas plant as a potential substrate for Pleurotus ostreatus full-scale cultivation. A preliminary in vitro trial shows that the mycelia can colonize at the same rate in both the conventional substrate, which was wheat straw added with sunflower cake and the experimental ones supplemented with corn digestate. The most important and innovative part of the work was full-scale cultivation. Quantitative analyses demonstrate that P. ostreatus is capable of growing on the conventional substrate as well as on the substrate containing 15% of digestate without reducing the mushroom harvest. Even better, digestate seems to stimulate a precocious and more homogeneous production. Chemical and spectroscopic analyses on the fruiting bodies confirm the results; in fact, mushrooms show a comparable structural composition, highlighted with FTIR and SERS spectroscopic evaluations. Moreover, the nutrient content did not change due to the addition of digestate. These promising results demonstrate that anaerobic digestate can become a resource for P. ostreatus cultivation without production losses and maintaining the same qualitative characteristics.