In this work, we provide a systematic study of CO2-water pretreatment of guayule biomass to optimize the residual ground bagasse from natural rubber extraction for hydrolysis and fermentation. Guayule biomass is mixed with water then loaded into a 250 mL reactor with exposure to a biphasic environment consisting of a CO2-rich vapor phase and water-rich liquid phase. The pressure is then rapidly released for a "physical expansion" effect. The pretreated biomass is enzymatically hydrolyzed, and the sugar concentration in hydrolysate is measured. Experimental runs are conducted in the temperature range of 145 to 210 degrees C and pressure range of 3.4 to 34 MPa. The solids ratio (dry solids mass/water mass) is between 0.17 and 1.7. The packing density is between 0.03 and 0.2 g of biomass per cm(3) of the reactor, and the holding time ranges from 20 to 840 min. A 4-fold increase in the reactor volume is performed and optimized with a 1-L vessel. High-pressure carbon dioxide-water pretreatment increases surface area of guayule biomass, introduces ruptured morphological features, and improves enzymatic digestibility. We achieve a total sugar yield of 85% (of theoretical) at two different reactor sizes of 250 mL and 1 L. At the smaller reactor, the optimum operational condition is 180 degrees C, 26 MPa, and 0.5 solid ratio. At the larger reactor, the optimum operational condition is 200 degrees C, 12 MPa, and 0.33 solid ratio.