For an alternative synthetic approach toward the heterocyclic industrial chemical ε-caprolactone, which is based on a biocatalytic oxidation of readily available cyclohexanol with air in aqueous media (using an alcohol dehydrogenase and a Baeyer–Villiger monooxygenase as enzyme components), a solvent engineering has been carried out identifying isooctane as a suitable co-solvent. Biotransformations in an aqueous-isooctane biphasic solvent system were found to proceed faster at both investigated substrate concentrations of 40 and 80 mm, respectively, compared with the analogous enzymatic reactions in pure aqueous medium. In addition, in all cases quantitative conversions were observed after a reaction time of 23 h when using isolated enzymes. The achievements indicate a high compatibility of isooctane [10%(v/v)] with the enzymes as well as the potential for an in situ removal of the organic reaction components, thus decreasing inhibition and/or destabilization effects of these organic components on the enzymes used. In contrast, so far, the use of recombinant whole-cells gave less satisfactory results, which might be due to limitations of the permeation of, for example, the substrate through the cell membrane.