In this work the influence of anaerobic fungi (AF) on biogas production has been studied. Strains of anaerobic fungi isolated from feces or rumen fluid of cows and deer were tested for their ability to integrate into anaerobic bacterial ecosystem, to improve degradation of substrate polysaccharides and consequently to influence biogas production. Batch culture, semicontinuous, and continuous experiments have been performed using anaerobic microbial consortium based on fermented pig slurry with different kind of substrates (celluloses, maize and grass silage), and different genera of anaerobic fungi. All experiments indicate positive effect of AF on biogas yield and quality. The biogas production was improved by anaerobic fungi by 5% up to 20%, methane ratio in biogas was higher about 2.5% depending on the used substrate and species of AF. Weak point of all cultivation experiments was inability of rumen fungi of long-term survival in fermenters with fermented pig slurry. The best results were achieved during continuous experiments with mixture of fungal cultures Anaeromyces (KF8) and Piromyces (KF9), where increased biogas production and quality was observed during the whole seven weeks experimental period.