The rise of antibiotic resistance demands the acceleration of molecular diversification strategies to inspire new chemical entities for antibiotic medicines. We report here on the large-scale engineering of ribosomally synthesized and post-translationally modified antimicrobial peptides carrying the ring-forming amino acid lanthionine. New-to-nature variants featuring distinct properties were obtained by combinatorial shuffling of peptide modules derived from 12 natural antimicrobial lanthipeptides and processing by a promiscuous post-translational modification machinery. For experimental characterization, we developed the nanoFleming, a miniaturized and parallelized high-throughput inhibition assay. On the basis of a hit set of >100 molecules, we identified variants with improved activity against pathogenic bacteria and shifted activity profiles, and extrapolated design guidelines that will simplify the identification of peptide-based anti-infectives in the future.