Emergence of vancomycin resistance in Gram-positive bacteria and the prevalence of vancomycin-resistant <i>Enterococci</i> (VRE) infections are highly alarming as very limited antibiotic options are available against VRE infections. Here, we present the synthesis of cholic acid-derived dimeric amphiphiles where two cholic acid moieties are tethered through carboxyl terminals using different alkylene spacers. Our investigations revealed that dimer <b>5</b> possessing a propylene spacer and glycine-valine peptides tethered on hydroxyl groups is the most effective antimicrobial against VRE. Dimer <b>5</b> can permeabilize bacterial membranes, generate reactive oxygen species, and clear preformed biofilms. We further demonstrate that dimer <b>5</b> downregulates vancomycin-mediated transcriptional activation of the vanHAX gene cluster and does not allow VSE to develop vancomycin resistance until 100 generations. Therefore, this study, for the first time, presents a bacterial membrane-targeting amphiphile that can mitigate VRE infections and inhibit the emergence of vancomycin resistance.