Theoretical calculations are used to explore a plausible mechanism for oxidative cleavage of cholesterol mediated by two ground-state O2 molecules. It is shown that cholesterol can form a stable pre-complex with the two triplet dioxygen molecules, which could be further stabilized in an enzyme environment by methionine (modelled here as Me2S). Triplet O2 can then react to form a metastable biradical species that is then further stabilized by reaction with a second triplet O2, resulting in an intermediate that undergoes an intersystem crossing to form a diperoxy intermediate. This in turn is reduced to the final cholesterol secosterol aldehyde product by the same methionine, which may provide an explanation for the presence of methionine sulfoxide fractions in Aβ amyloid peptide. The mechanistic theozyme model predicts an energetically viable pathway that is unusual in that triplet oxygen is normally considered to be unreactive in this context unless first excited to the singlet state. Although we show that the same reaction can also proceed via photosensitization of the complex if an appropriate cofactor is available, the energetics for the triplet oxygen reaction are competitive. Reactivity studies revealed that the reaction can also occur with other unsaturated substrates, with the lowest barriers occurring with more nucleophilic alkenes, or by rendering the 3O2 more electrophilic via non-covalent interactions with Me2S.