Abstract In this study, we investigated the in vitro efficacy of clarithromycin (CLA) combined with cefazolin (CFZ) or vancomycin (VCM) against Staphylococcus aureus biofilms formed on titanium devices in order to confirm the efficacy of eradication therapies against device-related infection. The distribution of CLA in muscle tissue surrounding bone was also investigated by liquid chromatography/tandem mass spectrometry in 10 orthopaedic patients. Biofilm formation and eradication of S. aureus were monitored by scanning electron microscopy and using double-staining dyes, respectively. Although S. aureus biofilms were not eradicated by CLA, CFZ or VCM alone, CLA combined with CFZ or VCM destroyed biofilms, and S. aureus eradication was clearly observed 72 h later. This in vitro study showed that treatment with CLA plus CFZ or VCM destroyed staphylococcal biofilms formed on medical devices and eradicated S. aureus.