Flavin-dependent halogenases are useful enzymes providing halogenated molecules with improved biological activity or intermediates for synthetic derivatization. Here we demonstrate how the fungal halogenase RadH can be used to regioselectively halogenate a range of bioactive aromatic scaffolds. Site-directed mutagenesis of RadH was used to identify catalytic residues and provide insights into the mechanism of fungal halogenases. A high throughput fluorescence screen was also developed enabling a RadH mutant to be evolved with improved properties. Finally we demonstrate how biosynthetic genes from fungi, bacteria and plants can be combined, to encode a new pathway generating a novel chlorinated coumarin ‘non-natural’ product in E. coli.