Streptomycete bacteria are a rich source of antibacterial natural products. Increasing antibiotic resistance is a global concern and novel classes of potent antibiotics are rare. Here we report the identification and genetic manipulation of the gene cluster for the cyclic antimicrobial peptide bottromycin in Streptomyces scabies. Bottromycin is active towards multi-drug resistant bacteria, such as MRSA and VRE, and contains a biologically unique macrocyclic amidine. The btm biosynthetic gene cluster was identified by genome mining and confirmed by genetic experiments. The metabolites of mutant strains were identified using liquid chromatography-mass spectrometry (LCMS), to characterise two radical SAM methyltransferases that are responsible for the β-methylation of three amino acids in bottromycin. A number of genes were also identified that are essential for bottromycin biosynthesis. A biosynthetic pathway has been proposed based on the results of these experiments coupled with bioinformatic analysis of the enzymes encoded in the btm cluster.