•Linezolid + rifampicin (RIF) was the best therapy for prosthetic joint infection by levofloxacin-resistant S. aureus.•Trimethoprim/sulfamethoxazole (SXT) + RIF did not prevent the emergence of RIF-resistant strains.•Concerns for clinical use of SXT + RIF should be further investigated. Combinations of linezolid (LZD) or trimethoprim/sulfamethoxazole (SXT) plus rifampicin (RIF) are alternative oral treatments for staphylococcal prosthetic joint infections (PJIs) when fluoroquinolones are not possible to use, but there is limited evidence regarding their activity. This study evaluated the efficacy of LZD and SXT, alone and in combination with RIF, against Staphylococcus aureus in an in vitro pharmacokinetic/pharmacodynamic biofilm model. Using the CDC Biofilm Reactor® system, simulated regimens of LZD (600 mg every 12 h), SXT (160/800 mg every 8 h) and levofloxacin (LVX) (750 mg/day), alone and in combination with RIF (600 mg/day), were evaluated against one methicillin-susceptible S. aureus (MSSA) and one methicillin-resistant S. aureus (MRSA) strain. Antibiotic efficacy was evaluated by the decrease in planktonic bacterial counts from medium and biofilm-embedded bacteria from coupons over 56 h. Resistant strains were screened. In both strains, SXT alone was ineffective and LZD presented low activity, but no resistance emerged. Combinations with RIF significantly increased the antibiofilm efficacy against MSSA (Δlog CFU/mL 56h–0h: SXT + RIF, −2.9 and LZD + RIF, −3.1), but RIF-resistant strains appeared with SXT + RIF. Against MRSA, LZD + RIF (−3.1) protected against the emergence of resistance and was more effective than SXT + RIF (−0.6; P < 0.05), in which RIF-resistant strains were again detected. LVX + RIF confirmed its high efficacy against biofilm-embedded bacteria, this being the most effective therapy (−5.1 against MSSA). The emergence of RIF-resistant strains with SXT + RIF poses serious concerns for its use in clinical practice. Interestingly, LZD + RIF appears to be an appropriate alternative for PJI caused by LVX-resistant S. aureus.