The increasing prevalence of multidrug-resistant (MDR) bacterial infections poses global health challenges, highlighting the urgent need for new antimicrobial agents. The key advantages of antimicrobial peptides are their ability to rapid bactericidal activity and their low propensity for resistance development. In this study, we designed a series of antimicrobial peptides by fusing the two fragments of antimicrobial peptides Cathelicidin-BF (1-9) and LL-37 (17-29), and then tested against selected Gram-negative and Gram-positive bacterial strains. Among these peptides, KF-22 displayed potent antibacterial activity against a panel of Gram-negative and Grampositive pathogens with MICs less than 5 mu g/mL, with demonstrating low toxicity. Moreover, KF-22 exhibits rapid bactericidal activity and a low propensity to induce resistance, simultaneously showing excellent antibiofilm and persisters activity. Mechanistic studies revealed that KF-22 induces membrane damage by targeting bacterial-specific membrane components, leading to the dissipate the proton motive force (PMF) and resulting in metabolic perturbations. Furthermore, in mice models no significant change was observed in plasma biochemical parameters between the treated and the control groups. In addition, in vivo studies confirmed that KF-22 are effective against drug-resistant pathogens. Taken together, the findings suggest that KF-22 is a promising candidate for further development to tackle MDR bacterial infections.