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Cotton stalks (CSs) are an abundant, renewable lignocellulose residue, which is usually burnt in the field to prevent propagation of vegetal diseases, causing economic losses and environmental concerns. The production of biogas has been considered as an alternative. This work aimed to improve the biogas production from CS by steam or organosolv plus supercritical carbon dioxide (scCO2) pre-treatment. All samples were pre-treated in a 500 mL autoclave for 140 min at 180 °C and fermented in 1 L eudiometer batch digesters for 42 days at 37.5 °C. The biogas and methane yields achieved from the untreated CS were 250 and 137 norm litres per kg of volatile solid (LN kg−1 VS), respectively. Pre-treatment of the CS samples with steam or the organosolv plus scCO2 process increased the methane yield by 20% compared to the untreated samples. The highest methane yield of 177 LN kg−1 VS was achieved by organosolv plus scCO2 pre-treatment at 100 bar and 180 °C for 140 min. Moreover, pre-treatment of the CS led to a significant reduction in the optimal digestion time from 30 days to 20 days for biogas production for the untreated CS.