In the presented datasets, we use newly developed isotopically labeled desthiobiotin azide (isoDTB) tags to residue-specifically quantify cysteines in a variety of bacterial strains and a human cell line with consistently high coverage. We e.g. quantify 59% of all cysteines in essential proteins in Staphylococcus aureus. We discover 88 cysteines with high reactivity, which correlates with functional importance. Furthermore, we identify 268 cysteines that are engaged by covalent ligands. Overall, a comprehensive map of the bacterial cysteinome is obtained.