Bacterial aminophenylpyrrole-derived alkaloids (APPAs) represent high value lead compounds. Pyrrolnitrin, which was developed into globally important fungicides, is the only reported APPA produced by Proteobacteria. Recently, various APPAs showing diverse bioactivities were discovered from Bacteroidetes. Here, a bioinformatics and phylogenetic approach enabled the elucidation of the biosynthesis of the highly diverse APPAs in Cytophagales bacteria and their chemical diversification strategy. The biosynthetic gene clusters were identified in producer strains, and the biosynthesis was experimentally validated by heterologous expression experiments in E. coli. First, one enzyme-dependent biosynthetic step yields the tryptophan-derived precursor 3-(2′-aminophenyl)-pyrrole. Second, a spontaneous Pictet-Spengler-like coupling reaction enables the bacterial producer strains to create a library of tricyclic alkaloids, since several aldehydes can be applied as substrates. The diversity of this natural products class is further enlarged by the catalytic action of a methyltransferase, which adds one or more methyl groups to the aminophenyl intermediate. © 2019 American Chemical Society.