The current management practice of digestate from biogas plants involves its use for land application as a fertilizer. Nevertheless, the inadequate handling of digestate may cause environmental risks due to losses of ammonia, methane and nitrous oxide. Therefore, the key goals of digestate management are to maximize its value by developing new digestate products, reducing its dependency on soil application and the consequent air pollution. The high nitrogen and lignin content in solid digestate make it a suitable substrate for edible and medicinal mushroom cultivation. To this aim, the mycelial growth rate and degradation capacity of the lignocellulosic component from corn silage digestate, undigested wheat straw and their mixture were investigated on Cyclocybe aegerita, Coprinus comatus, Morchella importuna, Pleurotus cornucopiae and Pleurotus ostreatus. The structural modification of the substrates was performed by using attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy. Preliminary in vitro results demonstrated the ability of P. ostreatus, P. cornucopiae and M. importuna to grow and decay hemicellulose and lignin of digestate. Cultivation trials were carried out on C. aegerita, P. cornucopiae and P. ostreatus. Pleurotus ostreatus showed the highest biological efficiency and fruiting body production in the presence of the digestate; moreover, P. ostreatus and P. cornucopiae were able to degrade the lignin. These results provide attractive perspectives both for more sustainable digestate management and for the improvement of mushroom cultivation efficiency.