The emergence of vancomycin-resistant bacteria has created an urgent need for new active analogues of vancomycin. We previously reported vancomycin dimers with in vivo antibacterial activity. Here, we provide the first experimental insights into their inhibitory actions in bacterial cell wall synthesis. New vancomycin derivatives 1 and 2 linked through the carboxylate terminals were prepared, along with previously reported dimers 3 and 4. Dimers 1–4 exhibited good-to-excellent activity against vancomycin-resistant enterococci (VRE) but 1 and 2 had significantly reduced activity against vancomycin-susceptible Staphylococcus aureus. Radio-isotope labeling experiments showed dimers 2 and 4 selectively inhibited peptidoglycan (PG) synthesis in VRE. In vitro cell wall synthesis assays using membrane fractions from S. aureus revealed that regardless of linking style, dimers inhibited both lipid intermediate (LI) and PG synthesis in both susceptible and resistant models, with more intensive inhibitory activity on PG synthesis in the resistant model. This provides the first experimental evidence that vancomycin dimers exhibit antibacterial activity against VRE through the suppression of cell wall synthesis.