AbstractGlutathione is not a universal coenzyme for formaldehyde oxidation. MySH (mycothiol, 1‐O‐(2′‐[N‐acetyl‐L‐cysteinyl]amido‐2′‐deoxy‐/propto‐D‐glucopyranosyl)‐D‐myo‐inositol) is GSH's counterpart as coenzyme in formaldehyde dehydrogenase from certain Gram‐positive bacteria. However, formaldehyde dissimilation and detoxification not only proceed via thiol‐dependent but also via thiol‐independent dehydrogenases. The distinct structures and enzymatic properties of MySH‐dependent and GSH‐dependent formaldehyde dehydrogenases could provide clues for development of selective drugs against pathogenic Mycobacteria. It is to be expected that other new types of thiol‐dependent formaldehyde dehydrogenases will be discovered in the future. Indications exist that the product of thiol‐dependent formaldehyde oxidation, the thiol formate ester, is not only hydrolytically converted into thiol and formate but can also be oxidatively converted in some cases by a molybdoprotein aldehyde dehydrogenase into the corresponding carbonate ester, decomposing spontaneously into CO2 and the thiol.