Considering the escalating challenge posed by antimicrobial resistance among Gram-negative bacteria, there is an urgent and critical imperative to explore and develop novel candidates for antimicrobial peptides (AMPs). The present study employed two strategies, lipid modification and amino acid substitution, to design a series of antimicrobial peptides (AMPs) based on the peptide WRK (WRLRWKTRWRLK). Among them, a peptide named FWK was identified to exhibit self-assembly behavior, forming nanoparticles of approximately 38 nm in size, ultimately leading to membrane destruction. Peptide FWK demonstrated rapid bactericidal activity against a multidrug-resistant (MDR) strain of Klebsiella pneumoniae within 30 min and maintained its efficacy for 36 h in vitro . In a mouse model of acute abdominal infection, peptide FWK demonstrated remarkable safety and potent antibacterial activity. A comparative analysis of various peptide modification strategies indicated that, in terms of antimicrobial activity, the positive charge and amphiphilicity of the peptide were more critical parameters than hydrophobicity. The substitution of amino acids offered a more versatile strategy for modification compared to lipid modification, thereby reducing the risk of introducing hemolytic toxicity. The antibacterial activity of the peptides was influenced by the type, quantity, and placement of substituted amino acids. The antibacterial activity can be enhanced by appropriately increasing the number of lysine and arginine residues. The biocompatibility of lysine was superior to that of arginine. The findings of this study offer valuable insights for the development of antimicrobial peptides (AMPs) that exhibit high efficacy and low toxicity, specifically targeting the effective management of infections caused by multidrug-resistant Gram-negative bacteria.