Bacterial contamination during the healing of bone defects frequently compromises the effects of bone regenerative therapy. Human beta-defensin-2 (hBD2) and -3 (hBD3) are antimicrobial peptides of human innate immune system with a broad antibacterial spectrum and rare bacterial resistance. The purpose of this study was to determine the effect of hBD2 and hBD3 on the healing of bacteria-contaminated bone defects. Rat bone marrow stromal cells (BMSCs) were infected with adenovirus to overexpress hBD2 or hBD3. Treatment with the conditioned medium derived from the BMSCs overexpressing defensins could concentration dependently reduce the viable Staphylococcus aureus numbers in the colony formation assay. In addition, the antimicrobial effect of BMSCs overexpressing defensins was verified with a diffusion chamber model in rats. Furthermore, we established a S. aureus-contaminated rat calvarial defect model and demonstrated that S. aureus contamination significantly compromised the bone regenerative effect after treatment with wild-type BMSCs. When defensin-overexpressing BMSCs were implanted into the S. aureus-contaminated defect, the viable S. aureus numbers were dramatically reduced and the negative effects of S. aureus contamination on bone healing were significantly mitigated. In conclusion, application of hBD2 or hBD3 promotes the healing of S. aureus-contaminated bone defects.