Ubiquinone (Coenzyme Q10, CoQ10), a yellow-to-orange-colored lipophilic substance having nutraceutical value, was extracted from dried biomass of Pseudomonas diminuta using supercritical carbon dioxide (SC-CO(2)). The effect of different operational parameters (temperature, pressure, and extraction time) and addition of co-solvent on SC-CO(2) extraction of CoQ10 was studied in detail. The solubility parameter of CoQ10, CO(2), and CO(2) with ethanol and methanol as co-solvents was calculated and validated with experimental results. Theoretically, ethanol and methanol had significant effect as co-solvent, and the difference between the two was only marginal. A maximum recovery of 22.33% was obtained using pure SC-CO(2) at 40 °C, 150 bar, and run time of 60 min. Ethanol as co-solvent at 3 mL/g of dried biomass at 40 °C and 150 bar increased the recovery from 22.33 to 68.57%. Further optimization of the extraction conditions by Box-Behnken design effectively increased the recovery to 96.2%. The optimized conditions were a temperature of 38 °C, pressure of 215 bar, and run time of 58 min.