Abstract Vancomycin is a critical last‐resort treatment for multidrug‐resistant Gram‐positive bacteria, particularly severe methicillin‐resistant S. aureus (MRSA) infections. However, the rise of vancomycin‐resistant strains significantly compromises its therapeutic efficacy. To overcome this challenge, recent research has focused on structural modifications of vancomycin using diverse strategies. Herein, the potential of a modification strategy by coupling vancomycin with antimicrobial peptides (AMPs) that have different mechanisms of action is explored. Among the acquired conjugates, Vm‐MSI showed a 20.18 fold improvement in antimicrobial activity over vancomycin and a 1.95 fold increase compared to the parent peptide MSI‐78. Vm‐MSI not only delays the development of resistance in vancomycin‐resistant S. aureus (VRSA) but also exhibits potent activity against a wide range of Gram‐negative bacteria. Additionally, Vm‐MSI demonstrated strong synergy with several conventional antibiotics of distinct mechanisms and displayed potent activities in eradicating biofilms and persisters. Mechanistic studies revealed the complex antibacterial mechanisms of Vm‐MSI, which hinder the development of bacterial resistance. In vivo, Vm‐MSI displayed remarkable therapeutic efficacy in mouse models of VRSA‐induced skin infection and multidrug‐resistant A. baumannii ‐induced lung infection. These findings underscore the great potential of Vm‐MSI as an effective treatment for infections caused by vancomycin‐resistant and Gram‐negative bacteria.