Decomposition of lignocellulosic plant biomass by four filamentous fungi was carried out to facilitate subsequent anaerobic degradation and biogas formation. Agricultural side products, wheat straw and corn stover and forestry energy plant willow chips were selected as plant biomass sources. The substrates were confronted by pure cultures of Penicillium aurantiogriseum (new isolate from rumen), Trichoderma reesei (DSM768), Gilbertella persicaria (SZMC11086) and Rhizomucor miehei (SZMC11005). In addition to total cellulolytic filter paper degradation activity, the production of endoglucanase, cellobiohydrolase, β-glucosidase enzymes were followed during the pretreatment period, which lasted for 10 days at 37 °C. The products of pretreatments were subsequently tested for mesophilic biogas production in batch reactors. All 4 strains effectively pretreated the lignocellulosic substrates albeit in varying degrees, which was related to the level of the tested hydrolytic enzyme activities. Penicillium aurantiogriseum showed outstanding hydrolytic enzyme production and highest biogas yield from the partially degraded substrates. Corn stover was the best substrate for biomass decomposition and biogas production. Scanning electron microscopy confirmed the deep penetration of fungal hyphae into the lignocellulosic substrate in all cases.