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Prenylated compounds are pharmacologically attractive natural products that are widely distributed in nature. Prenyltransferases (PTs) could catalyze the transfer of prenyl moieties to aromatic acceptors and increase the structural diversity and biological activity of natural products. In this work, a permissive O‐prenyltransferase AcaPT was discovered from Antrodia camphorata, a precious medicinal mushroom in Taiwan. AcaPT exhibited robust O‐prenylation capability toward structurally diverse drug‐like phenolic compounds, including antrodins, flavonoids, and coumarins. In total, 23 O‐prenylated products were obtained by scaled‐up enzymatic synthesis. AcaPT could be used as an efficient biocatalyst to synthesize O‐prenylated derivatives for drug discovery. [ABSTRACT FROM AUTHOR]