Biocatalytic reactions present an appealing substitute for conventional chemical pathways. Nonetheless, numerous biocatalytic reactions face constraints imposed by the enzyme's structural stability, leading to diminished yields and slower reaction rates. Given the experimental challenge of finding a suitable co-solvent mixture for the industry in time and resources, developing thermodynamic frameworks becomes essential to predict their impact on enzymatic and reaction properties. This work investigated the influence of co-solvent mixtures, temperature, and pressure on the enzymatic, kinetic and thermodynamic properties of two enzyme-catalyzed model reactions. First, for the oxidation of formate to carbon dioxide catalyzed by Candida boindinii formate dehydrogenase, the combined effect of pressure, temperature, and co-solvent mixtures on the kinetics and protein secondary structure was determined, finding a direct link between reaction rate and the enzyme structure. Second, co-solvent influence on reaction equilibrium was studied using ePC-SAFT, and on surface solvation using molecular dynamics for co-solvent accumulation. The experimentally observed co-solvent enhancement of the enzymatic activity is related to the compound's dynamics on the enzymatic surface. Thirdly, the effect of various mixtures of co-solvents at different concentrations on the structural stability of the enzyme was determined by unfolding assays. Fourth, co-solvent mixtures were tested in the oxidation of phenols catalyzed by Horseradish peroxidase, finding that enzymatic activity is directly related to the solvation energy of the liquid system. Therefore, this study highlights the significance of considering molecular methods to accurately depict the effect of co-solvent mixtures on enzymatic properties, encouraging the use of thermodynamic models to design enzyme-catalyzed processes.