Many antibacterial agents, including the glycopeptides, are inactive against Gram-negative bacteria because of their inability to cross the outer membrane of these cells. Different chemical and technological approaches have been described to circumvent such limitation. In this study, we aimed to apply the strategy of fusogenic liposomes, up to now used to carry biological compounds and materials inside cells, to localise a glycopeptide antibiotic, vancomycin (VAN), to the periplasmic space, thus allowing it to exert its bactericidal activity. Small unilamellar liposome vesicles were prepared by an extrusion procedure (SUVETs) from a phospholipid-cholesterol hemisuccinate mixture known for its fusogenic properties with the eukaryotic cell membrane. VAN was loaded with high efficiency into these vesicles and in microbiological experiments in vitro was shown to be able to inhibit to a different extent the growth of wild and standard Gram-negative bacterial strains. Minimum inhibitory concentrations as low as 6 mg/L were observed, for instance against clinical isolates of Escherichia coli and Acinetobacter baumannii. In comparison, neither the free antibiotic nor VAN-loaded 'classical' (non-fusogenic) liposomes showed any activity against the same bacteria. Scanning and transmission electron microscopy studies allowed confirmation that the produced SUVETs were able to adhere to and fuse with the external membrane of E. coli. According to preliminary experiments, this technological strategy can be proposed as a potentially successful way to enlarge the spectrum of activity of VAN.