Antimicrobial peptides (AMPs) have the potential to treat multidrug-resistant bacterial infections. However, the clinical application of AMPs is prevented by their toxicity and poor proteolytic stability. Here, a site-specific approach is used to generate new AMPs to improve their efficacy against bacterial pathogens while reducing their toxicity. We modified and generated a new series of antimicrobial peptides from the leucine- and lysine-rich antimicrobial peptide Amp1L (LKLLKKLLKKLLKLL) by the site-specific incorporation of an isopeptide bond while retaining the peptide's size, sequence, charge, and molecular weight. This single bond switch provides the peptides with a weak helical conformation, strong antimicrobial activity, resistance to proteolytic degradation, low toxicity, and lower hemolytic activity. This new site-specific approach offers a powerful tool for developing potent and nontoxic antimicrobial drugs.