Prenylation of cyclodipeptides contributes largely to the structure diversification and biological activity. The prenylated products can be further metabolized by modifications like hydroxylation with cytochrome P450 enzymes or nonheme FeII/2-oxoglutarate-dependent oxygenases. Herein, we cloned and overexpressed NFIA_045530 from Neosartorya fischeri, which shares high sequence similarity with the nonheme FeII/2-oxoglutarate-dependent oxygenase FtmOx1Af from Aspergillus fumigatus on the amino acid level. FtmOx1Af is a member of the biosynthetic enzymes for fumitremorgin-type mycotoxins and catalyzes the conversion of fumitremorgin B to verruculogen by insertion of an oxygen molecule into the two prenyl moieties. The recombinant protein EAW25734 encoded by NFIA_045530 was purified to apparent homogeneity and then was used for incubation with intermediates of the fumitremorgin biosynthetic pathway. LC-MS analysis revealed no consumption of fumitremorgin B but good conversion with its biosynthetic precursor tryprostatin B in the presence of FeII and 2-oxoglutarate. Structure elucidation confirmed 22-hydroxylisotryprostatin B and 14α, 22-dihydroxylisotryprostatin B as the major enzyme products. Further detailed biochemical characterization led to the identification of a novel enzyme, which catalyzes a double bond migration within the dimethylallyl moiety of tryprostatin B with concomitant hydroxylation. Incubation with 18O2-enriched atmosphere confirmed O2 as the major origin of the hydroxyl groups. Solvent exchange was also observed for that at C22. LC-MS analysis confirmed the presence of 22-hydroxylisotryprostatin B in a Neosartorya fischeri extract, highlighting the role of this enzyme in the metabolism of intermediates of the fumitremorgin/verruculogen pathway. A plausible reaction mechanism implementing a radical rearrangement prior to accepting a hydroxyl radical from FeIII is discussed.