Introduction: Combining currently available antibiotics to optimize their use is a promising strategy to reduce treatment failures against biofilm‐associated infections and new models must be developed to assess the efficacy of combinations in conditions similar to those encountered in patients. Material and Methods: We originally adapted a previously described hollow‐fiber (HF) infection model by allowing the formation of Staphylococcus aureus biofilm before drug exposure. We then mimicked different concentration profiles of amikacin and vancomycin similar to the free plasma concentration profiles observed in human patients treated daily over 5 days. We assessed the ability of both drugs, alone or in combination, to reduce planktonic and biofilm‐embedded bacteria and to prevent selection of resistance in both populations. Results and discussion: Although neither amikacin nor vancomycin alone had bactericidal activity on S. aureus, we identified a synergistic effect of their combination with a significant reduction of planktonic bacteria by −3.0 to −6.0 log10 CFU ml−1. In parallel, no obvious advantage of the combination compared to amikacin alone was observed on the biofilm‐embedded bacteria, the addition of vancomycin to amikacin only conferring a maximal reduction of 0.3 log10 CFU ml−1 more. We never found resistance to vancomycin whereas few bacteria less‐susceptible to amikacin were systematically present before treatment. We showed that these resistant bacteria, which were rapidly amplified by an exposure to amikacin alone, could be maintained at a low level in the biofilm population and even suppressed in the planktonic population by the addition of vancomycin. Conclusions: In conclusion, the adaptation of the HF model allowed us to demonstrate a differential bactericidal activity of vancomycin and amikacin combination on planktonic and biofilm‐embedded bacterial populations suggesting that the combination would not be much more efficacious than amikacin monotherapy for biofilm associated infections. However, the addition of vancomycin, by reducing the count of bacteria resistant to amikacin, could provide a relevant strategy to prevent the selection of antibiotic resistant bacteria during treatments. Due to its versatility, consisting in the possibility to simulate pharmacokinetic profiles of antibiotics in any species (animals, man), the HF model constitutes an invaluable tool to explore therapeutic strategies (monotherapy, combination) against biofilm‐associated infections of importance in veterinary medicine.