Bottromycin A2 is a structurally unique ribosomally synthesized and post-translationally modified peptide (RiPP) that possesses potent antibacterial activity towards multidrug-resistant bacteria. The structural novelty of bottromycin stems from its unprecedented macrocyclic amidine and rare β-methylated amino acid residues. The N-terminus of a precursor peptide (BtmD) is converted into bottromycin A2 by tailoring enzymes encoded in the btm gene cluster. However, little was known about key transformations in this pathway, including the unprecedented macrocyclization. To understand the pathway in detail, an untargeted metabolomic approach that harnesses mass spectral networking was used to assess the metabolomes of a series of pathway mutants. This analysis has yielded key information on the function of a variety of previously uncharacterized biosynthetic enzymes, including a YcaO domain protein and a partner protein that together catalyze the macrocyclization.