Binary mixtures of hydrophilic and hydrophobic solvents were assessed for their ability to balance enzyme activity with the conservation of enzyme stability in organic media. Acetone, dioxane and dodecane were chosen as model organic solvents, and subtilisin Carlsberg and horseradish peroxidase (HRP) were chosen as model enzymes. Residual enzyme activities were measured to monitor enzyme stability, and the fluorescence intensity of HRP was monitored to investigate structural changes due to the presence of an organic solvent. Enzyme stability increased with the increasing hydrophobicity of the solvent mixture used, and a solvent mixture with a high log P value (similar to > 4) was capable of conserving enzyme stability. Enzyme stability in organic media can be conserved therefore with a mixture of hydrophilic and hydrophobic solvents: this approach might be used as a general and practical strategy for optimizing enzyme activity and stability for industrial applications.