An understanding of the regulatory mechanisms that drive Staphylococcus aureus biofilm formation may lead to the development of an effective strategy to control the increasing number of refractory clinical infections it causes. The present study examined the effects of the antimicrobial agent human β‑defensin 3 (hBD‑3) and the antibiotics vancomycin and clindamycin on the expression of the S. aureus biofilm formation‑regulating genes, icaA and dltB, during bacterial adhesion and biofilm formation. Transcription (mRNA) levels of dlt and ica genes were measured using quantitative polymerase chain reaction, and slimes of S. aureus biofilm were examined with confocal scanning laser microscopy during S. aureus adhesion and biofilm formation. Although hBD‑3, vancomycin and clindamycin led to significantly attenuated biofilm formation, their treatment‑associated effects on the mRNA expression of dlt and ica were not identical. Vancomycin and clindamycin induced sustained expression of the dlt and ica genes, which may be harnessed to induce biofilm formation. However, hBD‑3 did not have a significant affect on the transcription level of dltB during either bacterial adhesion or biofilm formation. Therefore, the mechanism of hBD‑3 that regulated the suppression of biofilm formation appears to differ from the mechanisms of vancomycin and clindamycin.