Two Neocallimastbc frontalis strains, isolated from rumen fluid of a cow and of a chamois, were assessed for their ability to degrade lignocellulosic biomass. Two independent batch experiments were performed. Each experiment was split into two phases: hydrolysis phase and batch fermentation phase. The hydrolysis process during the N. frontalis incubation led to an initial increase of biogas production, an accelerated degradation of dry matter and an increased concentration of volatile fatty acids. As monitored by quantitative PCR, the applied N. frontalis strains were present and transcriptionally active during the hydrolysis phase but were fading during the batch fermentation phase. Thus, a separate hydrolytic pretreatment phase with anaerobic fungi, such as N. frontalis, represents a feasible strategy to improve biogas production from lignocellulosic substrates.