There is clearly a need for the development of new classes of antimicrobials to fight against multidrug-resistant bacteria. Here, we designed and synthesized of three ruthenium polypyridyl complexes: Ru(bpy)(BTPIP) (Ru(II)-1), Ru(bpy)(ETPIP) (Ru(II)-2) and Ru(bpy)(CAPIP) (Ru(II)-3) (N-N = bpy = 2,2'-bipyridine), their antimicrobial activities against S. aureus were assessed. The lead complexes of this set, Ru(II)-1(MIC = 0.016 mg/mL), was tested against biofilm. We also investigated whether bacteria can easily develop resistance to Ru(II)-1. The result demonstrated that S. aureus could not easily develop resistance to the ruthenium complexes. In addition, aimed to test whether ruthenium complexes treatment could increase the susceptibility of S. aureus to antibiotics, the synergism between Ru(II)-1 and common antibiotics against S. aureus were investigated using the checkerboard method. Interesting, Ru(II)-1 could increased the susceptibility of S. aureus to some aminoglycoside antibiotics(kanamycin and gentamicin). Finally, in vivo bacterial infection treatment studies were also conducted through murine skin infection model. These results confirmed ruthenium complexes have good antimicrobial activity in vitro and in vivo.