The emergence of multi-drug-resistant Gram-negative bacteria, including carbapenem-resistant Enterobacteriaceae, is a major health problem that necessitates the development of new antibiotics. Vancomycin inhibits cell-wall synthesis in Gram-positive bacteria but is generally ineffective against Gram-negative bacteria and is unable to penetrate the outer membrane barrier. In an effort to determine whether vancomycin and other antibiotics effective against Gram-positive bacteria could, through modification, be rendered effective against Gram-negative bacteria, we discovered that the covalent attachment of a single arginine to vancomycin yielded conjugates with order-of-magnitude improvements in activity against Gram-negative bacteria, including pathogenic E. coli. The vancomycin-arginine conjugate (V-R) exhibited efficacy against actively growing bacteria, induced the loss of rod cellular morphology, and resulted in the intracellular accumulation of peptidoglycan precursors, all consistent with cell-wall synthesis disruption as its mechanism of action. Membrane permeabilization studies demonstrated an enhanced outer membrane permeability of V-R as compared with vancomycin. The conjugate exhibited no mammalian cell toxicity or hemolytic activity in MTT and hemolysis assays. Our study introduces a new vancomycin derivative effective against Gram-negative bacteria and underscores the broader potential of generating new antibiotics through combined mode-of-action and synthesis-informed design studies.