The iron(IV)-oxo (ferryl) intermediate has been amply established as the principal oxidant in nonheme enzymes and the key player in C-H bond activations and functionalizations. In contrast to this status, our present QM/MM calculations of the mechanism of fosfomycin biosynthesis (a broad range antibiotic) by the nonheme HppE enzyme rule out the iron(IV)-oxo as the reactive species in the hydrogen abstraction (H-abstraction) step of the pro-R hydrogen from the (S)-2-hydroxypropylphosphonic substrate. Moreover, the study reveals that the ferryl species is bypassed in HppE, while the actual oxidant is an HO• radical hydrogen-bonded to a ferric-hydroxo complex, resulting via the homolytic dissociation of the hydrogen peroxide complex, Fe(II)-H2O2. The computed energy barrier of this pathway is 12.0 kcal/mol, in fair agreement with the experimental datum of 9.8 kcal/mol. An alternative mechanism involves the iron-complexed hydroxyl radical (FeIII-(HO•)) that is obtained by protonation of the iron(IV)-oxo group via the O-H group of the substrate. The barrier for this pathway, 23.0 kcal/mol, is higher than the one in the first mechanism. In both mechanisms, the HO• radical is highly selective; its H-abstraction leading to the final cis-fosfomycin product. It appears that HppE is prone to usage of HO• radicals for C-H bond activation, because the ferryl oxidant requires a specific H-abstraction trajectory (-FeOH ∼ 180°) that cannot be met for intramolecular H-abstraction. Thus, this work broadens the landscape of nonheme iron enzymes and makes a connection to Fenton chemistry, with implications on new potential biocatalysts that may harness hydroxyl radicals for C-H bond functionalizations. © 2016 American Chemical Society.