In the context of circular economy, cotton stalk constitutes an important agricultural residue, due to its high lignocellulosic, nitrogen, and phosphorus content, considering as valuable feedstock for various applications. However, lignified organic substances, such as wood, are not appropriate for anaerobic digestion, due to the complex structure of the lignocellulose and the low cellulolytic activity of the microbial community. Thus, hydrolysis of lignocellulose could be achieved by biomass pretreatment, implementing various approaches, including physicochemical, mechanical, and biological methods. In this study, a new biological pretreatment process was applied as an effective solution, in order to disrupt the lignocellulosic structure and enhance methane production during anaerobic digestion of cotton residues. Based on our data, methane production from the lignocellulosic biomass of cotton residues during this innovative biological pretreatment approach was estimated to be equal to 0.57 L CH4/g VS.d Taking into consideration that cotton stalk residues in Greece and in the EU are equal to 744,093 and 863,600 tones/year, their renewable energy potential is of high interest. The methane potential for energy and heating was measured by following the assumption that 1 m3 methane can yield 10 kWh during this innovative pretreatment method of cotton stalk. Based on the aforementioned yield, the exploitation of all quantities of cotton residues will result in the production of 347,607,737 and 403,436,185 Nm3 CH4/year in Greece and Europe respectively, which correspond to 3.4 million MWh/year in Greece and 4.0 million MWh/year in Europe. Assuming a nominal electrical efficiency of 35%, the generated electricity accounts for 12,166 MWhe/year in Greece and 14,120 MWhe/year in Europe. This represents a vital resource for renewable energy production in Europe, but also worldwide.