Enterobacteriaceae bacterial infections, particularly carbapenem-resistant Enterobacteriaceae (CRE) infections, pose a significant threat to global public health. With the rapid growth of antibiotic resistance, there is an urgent need to develop novel antimicrobial strategies. Antimicrobial peptides (AMPs), known as host defence peptides, are potentially potent alternatives to conventional antibiotics, given their broad spectrum of activity. In this study, we chemically modified Magainin 2, a type of AMP, with over 100 analogues by identifying the key active site through alanine scanning and incorporating hydrophobicity to promote antimicrobial activity. Our preliminary study showed several peptides exhibited significant antimicrobial activity against over 20 clinical isolates of Enterobacteriaceae strains. To further assess their applicability in complex infection environments, we demonstrated that the lead analogues can effectively inhibit biofilm formation, which is essential for controlling drug-resistant and chronic infections. Most importantly, the antimicrobial resistance induction experiments proved that long-term exposure to the lead analogues did not significantly induce resistance, suggesting a low risk of resistance in Enterobacteriaceae. Some of the peptides still showed superior inhibitory properties against pan-resistant (PDR) bacterial strains. Mechanistic studies showed that these peptides have a typical α-helix structure, which can achieve rapid bactericidal activity by disrupting the bacterial outer and inner membranes and increasing membrane permeability. Further evaluation showed that the peptides were stable in high-salt environments, trypsin and human plasma, and were biocompatible with low toxicity to mammalian cells. With the in vivo results pending, in this project, we have optimised Magainin 2-derived peptide with potent, broad-spectrum and stable antibacterial properties with low risk of drug resistance. Such lead candidates provide promising tools for multidrug-resistant bacterial infections, showing good prospects for clinical applications.