It is urgent to develop biocompatible and high-efficiency antimicrobial agents since microbial infections have always posed serious challenges to human health. Herein, through the marriage of facially amphiphilic skeletons and cationic dendrimers, high-density positively charged dendrimers D-CA6-N+ (G2) and D-CA2-N+ (G1) were designed and synthesized using the "branch" of facially amphiphilic bile acids, followed by their modification with quaternary ammonium charges. Both dendrimers could self-assemble into nanostructured micelles in aqueous solution. D-CA6-N+ displays potent antibacterial activity against Staphylococcus aureus and Escherichia coli, with minimum inhibitory concentrations (MICs) as low as 7.50 and 7.79 μM, respectively, and has an evidently stronger antibacterial activity than D-CA2-N+. Moreover, D-CA6-N+ can kill S. aureus faster than E. coli. The facial amphiphilicity of the bile acid skeleton facilitates the selective destruction of bacterial membranes and endows dendrimers with negligible hemolysis and cytotoxicity even under a high concentration of 16× MIC. In vivo studies show that D-CA6-N+ is much more effective and safer than penicillin G in treating S. aureus infection and promoting wound healing, which suggests facially amphiphilic skeleton-derived cationic dendrimers can be a promising approach to effectively enhance antibacterial activity and biocompatibility of antibacterial agent, simultaneously.