[Objective] To study the inhibition of staphylococcus aureus (S. aureus) biofilm by antimicrobial peptide 17BIPHE2 used alone or combined with antibiotics. [Methods] Congo red plate test and crystal violet staining test were used to assess the ability of biofilm formation by S. aureus. Minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of S. aureus were tested by broth microdilution method and agar plate method. The bacteria adhension was observed by S. aureus adhesion test and biofilm formation was analyzed by biofilm formation test. They were treated by 17BIPHE2 alone or combined with antibiotics. The removal of mature biofilm was observed by scanning electron microscope (SEM) after the treatment of 17BIPHE2 alone or combined with antibiotics. [Results] When MIC of 17BIPHE2 was 8 μmol/L, 1/2×MIC could inhibit the growth of planktonic bacteria effectively. In the adhension stage, the inhibition rate was 40% by 17BIPHE2 treated only. In the biofilm formation stage, the inhibition rate was 35%. Further, the rates for both stages decreased if combined with antibiotics. Antimicrobial peptide 17BIPHE2 could promote biofilm disintegration in the mature stage at 1/4×MIC concentration. Biofilm disintegration and bacterial adhesion could decrease at 1×MIC concentration. Furthermore, the combination of vancomycin promoted disintegration of bacterial biofilm as well as lots of leakage. [Conclusion] Antimicrobial peptides 17BIPHE2 was able to inhibit biofilm effectively, especially combined with antibiotics, could promote the inhibition more effectively. This article provides a new idea for treatment of S. aureus biofilm related infection.