The aim of the present study was to encapsulate vancomycin in different liposomal formulations and compare the antimicrobial activity against biofilms. Large unilamellar vesicles of conventional (LUV VAN), fusogenic (LUV VAN), and cationic (LUV VAN) liposomes encapsulating VAN were characterized in terms of size, polydispersity index, zeta potential, morphology, encapsulation efficiency (%EE) and release kinetics. The formulations were tested for their Minimum Inhibitory Concentration (MIC) and inhibitory activity on biofilm formation and viability, using methicillin-susceptible ATCC 29213 and methicillin-resistant ATCC 43300 strains. LUV VAN showed better %EE (32.5%) and sustained release than LUV VAN, LUV VAN, and free VAN. The formulations were stable over 180 days at 4°C, except for LUV VAN, which was stable up to 120 days. The MIC values for liposomal formulations and free VAN ranged from 0.78 to 1.56 µg/ml against both tested strains, with no difference in the inhibition of biofilm formation as compared to free VAN. However, when treating mature biofilm, encapsulated LUV VAN increased the antimicrobial efficacy as compared to the other liposomal formulations and to free VAN, demonstrating a better ability to penetrate the biofilm. Vancomycin encapsulated in fusogenic liposomes demonstrated enhanced antimicrobial activity against mature biofilms.