Susceptibility to antibiotics is dramatically reduced when bacteria form biofilms. In clinical settings this has a profound impact on treatment of implant-associated infections, as these are characterized by biofilm formation. Current routine susceptibility testing of microorganisms from infected implants does not reflect the actual susceptibility, and the optimal antibiotic strategy for treating implant-associated infections is not established. In this study of biofilm formation in implant-associated osteomyelitis, we compared the in vitro and in vivo efficacy of selected antibiotics alone and in combination against Staphylococcus aureus. We tested vancomycin, linezolid, daptomycin and tigecycline alone and in combination with rifampicin, vancomycin, linezolid and daptomycin against S. aureus. In vitro, biofilm formation dramatically reduced susceptibility by a factor of 500–2000. In vivo, antibiotic combinations were tested in a murine model of implant-associated osteomyelitis. Mice were infected by inserting implants colonized with S. aureus trough their tibia. After 11 days, the animals were divided into different groups (five animals/group) and given 14 days of antibiotic therapy. All antibiotics resulted in a reduced bacterial load in the infected bone surrounding the implant. Overall, the most effective antibiotic combinations contained rifampicin. Combinations containing two non-rifampicin antibiotics were not more active than single drugs.