Prenylated flavin mononucleotide (prFMN) is a recently discovered modified flavin cofactor containing an additional nonaromatic ring, connected to the N5 and C6 atoms. This cofactor underpins reversible decarboxylation catalyzed by members of the widespread UbiD enzyme family and is produced by the flavin prenyltransferase UbiX. Oxidative maturation of the UbiX product prFMNH2 to the corresponding oxidized prFMNiminium is required for ferulic acid decarboxylase (Fdc1; a UbiD-type enzyme) activity. However, it is unclear what role the Fdc1 enzyme plays in this process. Here, we demonstrate that, in the absence of Fdc1, prFMNH2 oxidation by O2 proceeds via a transient semiquinone prFMNradical species and culminates in a remarkably stable prFMN-hydroperoxide species. Neither forms of prFMN are able to support Fdc1 activity. Instead, enzyme activation using O2-mediated oxidation requires prFMNH2 binding prior to oxygen exposure, confirming that UbiD enzymes play a role in O2-mediated oxidative maturation. In marked contrast, alternative oxidants such as potassium ferricyanide support prFMNiminium formation both in solution and in Fdc1.