Hydrothermal pretreatment of five lignocellulosic substrates (i.e. wheat straw, rice straw, biomass sorghum, corn stover and Douglas fir bark) were conducted in the presence of CO2 as a catalyst. To maximize disintegration and conversion into bioenergy (methane and hydrogen), pretreatment temperatures and subsequent pressures varied with a range of 26–175 °C, and 25–102 bars, respectively. Among lignin, cellulose and hemicelluloses, hydrothermal pretreatment caused the highest reduction (23–42%) in hemicelluloses while delignification was limited to only 0–12%. These reductions in structural integrity resulted in 20–30% faster hydrolysis rates during anaerobic digestion for the pretreated substrates of straws, sorghum, and corn stover while Douglas fir bark yielded 172% faster hydrolysis/digestion due to its highly refractory nature in the control. Furans and phenolic compounds formed in the pretreated hydrolyzates were below the inhibitory levels for methane and hydrogen production which had a range of 98–340 ml CH4/g volatile solids (VS) and 5–26 ml H2/g VS, respectively. Results indicated that hydrothermal pretreatment is able to accelerate the rate of biodegradation without generating high levels of inhibitory compounds while showing no discernible effect on ultimate biodegradation.