Lipophilic substitution on vancomycin is an effective strategy for the development of novel vancomycin analogues against drug-resistant bacteria by enhancing bacterial cell wall interactions. However, hydrophobic structures usually lead to long elimination half-life and accumulative toxicity; therefore, hydrophilic fragments were also introduced to the lipo-vancomycin to regulate their pharmacokinetic/pharmacodynamic properties. Here, we synthesized a series of new vancomycin analogues carrying various sugar moieties on the seventh-amino acid phenyl ring and lipophilic substitutions on vancosamine with extensive structure-activity relationship analysis. The optimal analogues indicated 128-1024-fold higher activity against methicillin-susceptible S. aureus, vancomycin-intermediate resistant S. aureus (VISA), and vancomycin-resistant Enterococci (VRE) compared with that of vancomycin. In vivo pharmacokinetics studies demonstrated the effective regulation of extra sugar motifs, which shortened the half-life and addressed concerns of accumulative toxicity of lipo-vancomycin. This work presents an effective strategy for lipo-vancomycin derivative design by introducing extra sugars, which leads to better antibiotic-like properties of enhanced efficacy, optimal pharmacokinetics, and lower toxicity.