The enhancement of the yields of the anaerobic digestion process was the primary target of the present study, thanks to the development of an innovative fungal pretreatment. The fungal community of agrozootechnical biogas plant feedstocks and by-products (i.e., maize silage and stover, zootechnical slurry and manure, wheat and rice straw, and solid digestate) were studied, isolating and identifying 172 strains belonging to 89 taxa. The mycobiota was dependent on the biomass: maize and zootechnical samples showed a higher biodiversity than straw and solid digestate. During the preliminary screening, Cephalotrichum stemonitis extensively grew on both non-sterile maize silage and solid digestate and it was then selected for their pretreatment. The fungus did change the biomasses features: the total solids decreased (approximately 20%), while the volatile solids slightly changed (<1%). Lignin, cellulose and hemicellulose degradation rate in maize silage was higher (55.2%, 25.0% and 24.5%, respectively) than in solid digestate (8.7%, 0.6% and 10.9%, respectively), highlighting their different accessibility and recalcitrance. During the anaerobic digestion, fungal-pretreated maize silage produced lower cumulative biogas and methane than the control, probably due to an excessive degradation of easily accessible fibers. On the contrary, the pretreated solid digestate was less recalcitrant to the microbial transformation in the anaerobic digestion: as a result, cumulative biogas and methane yields were significantly enhanced (approximately 70%). By favoring the (re)use of digestate as a feedstock, the investigated fungal pretreatment could help the economic and environmental sustainability of the anaerobic digestion.