Sugarcane juice is a nutritious and energetic drink. For its processing, the use of supercritical carbon dioxide (SC-CO2) technology as an intervention potentially capable of rendering a high quality product can be considered. This study evaluated the combined effect of SC-CO2 and mild temperatures, primarily aiming for the reduction of endogenous microorganisms and enzymes in sugarcane juice (pH~5.5). Pressures (P) ranging from 74 to 351 bar, temperatures (T) between 33 and 67 °C, and holding times (t) between 20 and 70 min were tested in a central composite rotational design. Seventeen trials were performed, comprising three replicates at the central points. Counts of aerobic mesophiles, molds and yeasts, lactic acid bacteria and coliforms at 45 °C, determination of polyphenol oxidase (PPO) and peroxidase (POD) activities, and measurement of color parameters in freshly extracted and processed juice’s samples were carried out. The pH of fresh and processed juice varied between 4.6 and 6.0, and between 4.6 and 6.3, respectively. The number of decimal reductions achieved in mesophiles, molds and yeasts, lactic acid bacteria and coliforms varied between 0.1 and 3.9, 2.1 and 4.1, 0.0 and 2.1, and 0.3 to 2.5, respectively. The percentages of PPO reduction ranged from 3.51% to 64.18%. Regarding the POD, reductions between 0.27% and 41.42% were obtained. Color variations between fresh and processed samples varied between 2.0 and 12.3. As for mesophiles, molds and yeasts reduction, and soluble solids variation, none of the variables or their interactions were significant. In terms of polyphenol oxidase (PPO) reduction, only t was significant; however, T, t, and the interaction between them significantly affected the peroxidase (POD) reduction. In regards to pH variation, P, and the interaction between T and t were significant. P, T, t, and the interaction between T and t played a significant effect on color. The combination of mild temperatures and SC-CO2 can be potentially used for cane juice preservation.