Prenylated FMN (prFMN) is a recently discovered cofactor required by the UbiD family of reversible decarboxylases involved in ubiquinone biosynthesis and in the biotransformation of aromatic compounds. This cofactor is synthesized by UbiX-like prenyltransferases catalyzing the transfer of the dimethylallyl moiety of dimethylallyl-monophosphate (DMAP) to FMN. The origin of DMAP for prFMN biosynthesis and the biochemical properties of free prFMN are unknown. We show that in Escherichia coli cells, DMAP can be produced by phosphorylating prenol using ThiM or dephosphorylating DMAPP using Nudix hydrolases. We produced 14 active prenyltransferases whose properties enabled the production of protein-free forms of prFMN. In vitro assays revealed that the UbiD-like ferulate decarboxylase (Fdc1) has high affinity to free prFMNiminium and C1′-ene-prFMNiminium and can be activated under both oxidized and reduced conditions. These insights into the biosynthesis and properties of prFMN will facilitate further elucidation of the biochemical diversity of reversible UbiD (de)carboxylases.