Biocatalysis can be applied in aqueous media and in different non-aqueous solutions (non-conventional media). Water is a safe solvent, yet many synthesis-wise interesting substrates cannot be dissolved in aqueous solutions, and thus low concentrations are often applied. Conversely, non-conventional media may enable higher substrate loadings but at the cost of using (fossil-based) organic solvents. This paper determines the CO2 production—expressed as kg CO2·kg product−1—of generic biotransformations in water and non-conventional media, assessing both the upstream and the downstream. The key to reaching a diminished environmental footprint is the type of wastewater treatment to be implemented. If the used chemicals enable a conventional (mild) wastewater treatment, the production of CO2 is limited. If other (pre)treatments for the wastewater are needed to eliminate hazardous chemicals and solvents, higher environmental impacts can be expected (based on CO2 production). Water media for biocatalysis are more sustainable during the upstream unit—the biocatalytic step—than non-conventional systems. However, processes with aqueous media often need to incorporate extractive solvents during the downstream processing. Both strategies result in comparable CO2 production if extractive solvents are recycled at least 1–2 times. Under these conditions, a generic industrial biotransformation at 100 g L−1 loading would produce 15–25 kg CO2·kg product−1 regardless of the applied media. [ABSTRACT FROM AUTHOR]