The aim of this research was to develop a method for pretreatment of lignocellulose (LC) substrates (harvest residues (HR)) via electroporation (EP) for the purpose of improving the biogas production process. In addition, pretreated LC substrates were analyzed by scanning electron microscopy (SEM) and the energy balance of the total process was calculated. After the conducted pretreatment and anaerobic co-digestion with dairy cow manure (DCM), the statistical data analysis showed statistically significant differences in biogas and/or methane yield for all three LC substrates and their fractions. It was concluded that, after the pretreatment of LC substrates via EP, it is possible to improve the anaerobic co-digestion process and to achieve positive energy balance of the total process.