Valorization of lignocellulosic biomass, such as Spent Mushroom Substrate (SMS), as an alternative substrate for biogas production could meet the increasing demand for energy. In view of this, the present study aimed at the biotechnological valorization of SMS for biogas production. In the first part of the study, two SMS chemical pretreatment processes were investigated and subsequently combined with thermal treatment of the mentioned waste streams. The acidic chemical hydrolysate derived from the hydrothermal treatment, which yielded in the highest concentration of free sugars (≈36 g/100 g dry SMS, hydrolysis yield ≈75% w/w of holocellulose), was used as a potential feedstock for biomethane production in a laboratory bench-scale improvised digester, and 52 L biogas/kg of volatile solids (VS) containing 65% methane were produced in a 15-day trial of anaerobic digestion. As regards the alkaline hydrolysate, it was like a pulp due to the lignocellulosic matrix disruption, without releasing additional sugars, and the biogas production was delayed for several days. The biogas yield value was 37 L/kg VS, and the methane content was 62%. Based on these results, it can be concluded that SMS can be valorized as an alternative medium employed for anaerobic digestion when pretreated with both chemical and hydrothermal hydrolysis.