Antibiotic resistance (ABR) is a naturally occurring phenomenon that is being exacerbated by inappropriate use of antibiotics to treat human infections. Infections caused by resistant pathogens lead to more severe, long-term illness or fatality. As ABR becomes more prevalent, the development of novel antibiotics is necessary to overcome resistant infections. Antimicrobial peptides (AMPs) provide an untapped source of potential antibacterial therapeutics. Of particular interest are bacteriocins, AMPs produced by bacteria, which exhibit bacteriostatic or bactericidal activity. The circularized bacteriocin enterocin AS-48 produced by Enterococcus sp. exhibits antibacterial activity through untargeted membrane disruption. The membrane-penetrating activity of enterocin AS-48 has been attributed to a specific alpha-helical region on the circular peptide. Truncated, linearized forms containing these domains have been shown to preserve limited bactericidal activity. I utilized the amino acid sequence of the active helical domain of enterocin AS-48 to perform a homology-based search of similar sequences in other bacterial genomes. I identified similar domains in three previously uncharacterized AS-48-like bacteriocin genes in Clostridium sordellii, Paenibacillus larvae, and Bacillus xiamenensis. Enterocin AS-48 and AS-48-like homologs from these bacterial species were used as scaffolds for the design of minimal peptide libraries based on the active helical domain of each bacteriocin sequence. Rational design techniques took into account charge, hydrophobicity, and amphipathicity to create novel AMP libraries. In total, 95 synthetic peptide variants of each scaffold peptide, designated Syn-enterocin, Syn-sordellicin, Syn-larvacin, and Syn-xiamencin, were designed and synthesized from each scaffold sequence based on defined biophysical parameters. A total of 384 total peptides were assessed for antibacterial activity against a panel of Gram-negative and Gram-positive bacteria. Minimal Inhibitory Concentrations (MICs) were observed as low as 15.6 nM to Escherichia coli, 125 nM to Streptococcus pyogenes and 250 nM to multidrug-resistant Staphylococcus aureus (MRSA), with no significant cytotoxicity to eukaryotic cells. Characterization of secondary structure and evaluation of potential mode of action of our most potent peptide variants suggest an alpha-helical structure causing bacterial membrane disruption. Further, peptide drug candidates were evaluated for potential to develop resistance in E. coli to evaluate long-term, therapeutic use preservation. Bacterial colonies with stable mutations that caused resistance to peptide drug candidates were isolated. Six resistant mutants were observed to be cross resistant with cell wall inhibiting antibiotics, however, this resulted in decreased bacterial fitness. It was determined that changes to O-antigen chain length, shifts in lipid A asymmetry, and increased net charge of lipid A caused changes to susceptibility to the peptide drug candidates. My work demonstrates for the first time a general workflow of using minimal domains of natural bacteriocin sequences as scaffolds to design and rapidly synthesize a library of bacteriocin-based antimicrobial peptide variants for evaluation.