Developing sustainable biocatalytic processes requires alternative solvents that support enzyme activity while reducing environmental impact. This study explores the potential to use diformylxylose (DFX), a xylose-derived green solvent, as a cosolvent in enzymatic reactions, and compares its application to reaction outcomes in conventional solvents such as dimethyl sulfoxide (DMSO) and dimethylformamide (DMF). A comprehensive enzyme panel, including ketoreductases (KREDs), lipases as well as transaminases (TAs) and imine reductases (IREDs) was tested for activity and stability in DFX. In the green solvent, the selected KREDs and the immobilized lipase CalB retained high or even superior catalytic activity compared to conventional media, while the selected biocatalysts from other enzyme classes such as TAs, and IREDs exhibited limited compatibility under the tested conditions underscoring the enzyme-specific nature of solvent effects. Notably, the KRED TeSADH W110A achieved full conversion when asymmetrically reducing phenyl-ring-containing ketones at 300 mM substrate concentration in DFX, significantly outperforming reaction conditions with DMSO and DMF (∼40% conversion). Lipase CalB also exhibited remarkable activity, reaching 95% conversion at 300 mM 4-nitrophenyl butyrate loading. The findings highlight DFX as a promising alternative solvent for biocatalysis applications, particularly for KRED- and lipase-mediated reactions.