The emergence of multidrug-resistant (MDR) pathogens has become a global public health crisis. Among them, MDR is the main cause of nosocomial infections and deaths. Antimicrobial peptides (AMPs) are considered as competitive drug candidates to address this threat. In the study, we characterized two AMPs (AS-hepc3 and AS-hepc3) that had potent activity against 5 new clinical isolates of MDR . Both AMPs destroyed the integrity of the cell membrane, induced leakage of intracellular components, and ultimately led to cell death. A long-term comparative study on the bacterial resistance treated with AS-hepc3, AS-hepc3 and 12 commonly used antibiotics showed that quickly developed resistance to the nine antibiotics tested (including aztreonam, ceftazidime, cefepime, imipenem, meropenem, ciprofloxacin, levofloxacin, gentamicin, and piperacillin) as early as 12 days after 150 days of successive culture generations. The initial effective concentration of 9 antibiotics against was greatly increased to a different high level at 150 days, however, both AS-hepc3 and AS-hepc3 maintained their initial MIC unchangeable through 150 days, indicating that did not produce any significant resistance to both AMPs. Furthermore, AS-hepc3 did not show any toxic effect on mammalian cells and mice . AS-hepc3 had a therapeutic effect on MDR infection using a mouse lung infection model and could effectively increase the survival rate of mice by inhibiting bacterial proliferation and attenuating lung inflammation. Taken together, the short peptide AS-hepc3 would be a promising agent for clinical treatment of MDR infections.