Infections caused by vancomycin-resistant enterococci (VRE) are increasing in most countries, including Germany. As there are very few therapeutic options, the development of novel compounds that overcome resistant phenotypes is urgently needed. Here, we report the design, synthesis and activity testing of vancomycin-peptide conjugates that are highly active against a broad range of Gram-positive pathogens, including VRE, MRSA and Clostridioides difficile. The most active conjugate, a hexaarginine-vancomycin conjugate designated FU002, was found to be highly active against all resistant pathogens tested, with the highest activity against vanB E. faecium strains. The compound has bactericidal activity, killing vancomycin resistant strains within four hours of incubation. In vivo studies in Galleria melonella larvae and murine systemic infection models demonstrated efficacy as indicated by increased survival of G. melonella larvae and reduced colony forming unit counts in organs of mice treated with FU002. The mode of action of the peptide-vancomycin conjugates differs from that of vancomycin as shown by transcriptome studies. In conclusion, vancomycin-peptide conjugates represent a promising class of novel antibiotic compounds against highly resistant Gram-positive pathogens.