Dioxygen (O2) is a potent oxidant used by aerobic organisms for energy transduction and critical biosynthetic processes. Numerous metalloenzymes harness O2 to mediate C-H bond hydroxylation reactions, but most commonly feature iron or copper ions in their active site cofactors. In contrast, many manganese-activated enzymes─such as glutamine synthetase and isocitrate lyase─perform redox neutral chemical transformations and very few are known to activate O2 or C-H bonds. Here, we report that the dimanganese-metalated form of the cambialistic monooxygenase SfbO (Mn2-SfbO) can efficiently mediate enzymatic C-H bond hydroxylation. The activity of the dimanganese form of SfbO toward substrate hydroxylation is comparable to that of its heterobimetallic Mn/Fe form but exhibits distinct kinetic profiles. Kinetic, spectroscopic, and structural studies invoke a mixed-valent dimanganese cofactor (MnIIMnIII) in O2 activation and evidence a stoichiometric role for superoxide in maturing an O2-inert MnII2 cofactor. Computational studies support a hypothesis wherein superoxide addition to the MnII2 cofactor installs a critical bridging hydroxide ligand that stabilizes higher-valent manganese oxidation states. These findings establish the viability of proteinaceous dimanganese cofactors in mediating complex, multistep redox transformations.