Today's increasing demand for sustainable technologies and circular production systems promotes the development of R&D and innovations for green, renewable, and economically viable solutions. Biomass utilization has a great opportunity in this respect, because of its flexible applicability. This study aimed to investigate the effects of fungal pretreatment on lignocellulose substrate for semi-continuous anaerobic digestion. The applied substrate was an I-214 poplar clone, and biological pretreatment was carried out with Pleurotus strain HK35. An intensive organic loading rate was applied in order to compare the digestion parameters between the treated and control fermenters. Results showed significant benefits in the case of daily methane yield and specific methane yield, as well (205 mLd-1L-1, 44 mLgVS-1L-1 in case of fungal pretreated and 98 mLd-1L-1, 26 mLgVS-1L-1 in case of control fermenters). Titrated volatile fatty acid content indicated stable run, cellulose and reduced lignin content demonstrated the efficiency of degradation, supporting the accelerated methane production.