Cytochrome P450s catalyze a diverse array of chemical transformations that are essential for biosynthesis and metabolic processes. While the ferryl-oxo heme radical cation Compound I (Cpd I) has been accepted to be the principal oxidant, the Fe(III)–H2O2 complex has been widely proposed to be an alternative oxidant for sulfoxidation in P450s. However, few definitive evidences have been presented to either confirm or rule out the role of Fe(III)–H2O2 as the active species in P450s. Herein, we revisited this long-standing issue in different variants of CYP199A4. For the T252E mutant, a quantum mechanical and molecular mechanical (QM/MM) study suggests that the H2O2 activation is dominated by the E252-mediated O–O heterolysis mechanism, leading to the Cpd I species for sulfoxidation. Instead, the FeIII(H2O2) complex acts as the oxidant for the direct sulfoxidation in T252A, bypassing the active species of Cpd I. Further experiments enable us to identify the T252Q mutant that has comparable activity as T252E. In contrast to T252E, the QM/MM study suggests that Fe(III)–H2O2 functions as an efficient oxidant for sulfoxidation in the T252Q mutant, bypassing the formation of Cpd I. Thus, this work not only reveals a “two-oxidant” scenario for the sulfoxidation reaction in P450s but also highlights an alternative mechanistic pathway that may avoid the irreversible inactivation of P450s caused by interactions between Cpd I and excess H2O2 molecules.