Rattusin, an α-defensin-related antimicrobial peptide isolated from the small intestine of rats, has been previously characterized through NMR spectroscopy to elucidate its three-dimensional structure, revealing a C2 homodimeric scaffold stabilized by five disulfide bonds. This study aimed to identify the functional region of rattusin by designing and synthesizing various short analogs, subsequently leading to the development of novel peptide-based antibiotics. The analogs, designated as F1, F2, F3, and F4, were constructed based on the three-dimensional configuration of rattusin, among which F2 is the shortest peptide and exhibited superior antimicrobial efficacy compared to the wild-type peptide. The central cysteine residue of F2 prompted an investigation into its potential to form a dimer at neutral pH, which is critical for its antimicrobial function. This activity was abolished upon the substitution of the cysteine residue with serine, indicating the necessity of dimerization for antimicrobial action. Further, we synthesized β-hairpin-like analogs, both parallel and antiparallel, based on the dimeric structure of F2, which maintained comparable antimicrobial potency. In contrast to rattusin, which acts by disrupting bacterial membranes, the F2 dimer binds directly to DNA, as evidenced by fluorescence assays and DNA retardation experiments. Importantly, F2 exhibited negligible cytotoxicity up to 515 μg/mL, assessed via hemolysis and MTT assays, underscoring its potential as a lead compound for novel peptide-based antibiotic development.