The influence of the nitrogen fertilization level was investigated relative to the chemical composition of lignocellulosic energy crops and their usefulness as a substrate for the purpose of biogas production. In the case of perennial crops, such as Virginia mallow (VM) and reed canary grass (RCG), the impacts of individual swath and cutting frequency were examined. The results showed that raised nitrogen fertilization improved the biomass quality. This was important for biogas production, primarily through decreased lignin content, and for an increased ratio of structural carbohydrates to lignin. It is believed that this tendency may facilitate the digestion of the tested substrate and increase the methane fermentation efficiency. Likewise, the swath of perennial crops differed significantly in terms of the analyzed properties, which also may have been reflected in the suitability of biomass as a feedstock for biogas plants.