Bacteriocins hold unprecedented promise as a largely untapped source of antibiotic alternatives in the age of multidrug resistance. Here， we describe the first approach to systematically design variants of a novel AS-48 bacteriocin homologue， which we have termed safencin AS-48， from Bacillus safensis， to gain insights into engineering improved activity of bacteriocins. A library of synthetic peptides in which systematic amino acid substitutions to vary the periodicity and abundance of polar， acidic， aliphatic， and hydrophobic residues were generated for a total of 96 novel peptide variants of a single bacteriocin candidate. Using this method， we identified nine synthetic safencin (syn-safencin) variants with broad and potent antimicrobial activities with minimal inhibitory concentrations (MIC) as low as 250 nM against E. coli， P. aeruginosa， X. axonopodis， and S. pyogenes with minimal cytotoxicity to mammalian cells. It is anticipated that the strategies we have developed will serve as general guides for tuning the specificity of a given natural bacteriocin compound for therapeutic specificity.