New strategies are urgently needed to target MRSA, a major global health problem and the leading cause of mortality from antibiotic-resistant infections in many countries. Here, we report a general approach to this problem exemplified by the design and synthesis of a vancomycin-D-octaarginine conjugate (V-r8) and investigation of its efficacy in addressing antibiotic-insensitive bacterial populations. V-r8 eradicated MRSA biofilm and persister cells in vitro, outperforming vancomycin by orders of magnitude. It also eliminated 97% of biofilm-associated MRSA in a murine wound infection model and displayed no acute dermal toxicity. This new dual-function conjugate displays enhanced cellular accumulation and membrane perturbation as compared to vancomycin. Based on its rapid and potent activity against biofilm and persister cells, V-r8 is a promising agent against clinical MRSA infections.