Microbial enzymes can perform reactions that remain out of reach for traditional organic chemists. Harnessing the full diversity of microbial chemistry could improve how chemists and metabolic engineers access small molecules, including important drugs. We are discovering functionally unique enzymes by investigating how microbes make structurally unusual natural products. Using an understanding of biosynthetic logic, we identify natural products containing functional groups and bond constructions that cannot be explained using known enzymatic chemistry. Identification of the cylindrocyclophane, lomaiviticin, bartoloside, and cremeomycin biosynthetic gene clusters and their encoded enzymes has revealed intriguing reactivity, including enzymes that halogenate unactivated carbon centers, link aromatic rings with alkyl halides, and construct N–N bonds. In addition to enriching our fundamental knowledge of enzymatic catalysis, these enzymes will expand the ‘toolkit’ available for biocatalysis and metabolic engineering efforts and fill critical needs in chemical synthesis.