The combination of antimicrobial agents has been proposed as a therapeutic strategy to control bacterial diseases and to reduce the emergence of antibiotic-resistant strains in clinical environments. In this study, the interaction between the lantibiotic bovicin HC5 with chloramphenicol, gentamicin, nisin, lysostaphin and hydrogen peroxide against Staphylococcus aureus O46 was evaluated by MIC assays. The central composite rotatable design (CCRD), a robust and economic statistical design, was used to combine concentration levels of different antimicrobials agents with distinct mechanisms of action and the presence of significant interactions among the antimicrobials was determined by regression analysis. According to the adjusted model, there were no significant interactions between bovicin HC5 and gentamicin, lysostaphin, nisin or hydrogen peroxide. However, bovicin HC5 showed a significant interaction (P < 0.02) with chloramphenicol. This is the first study applying the CCRD approach to evaluate the combined effect of antimicrobials against S. aureus. Based on our results, this approach is an effective strategy to determine synergistic interactions between antimicrobial agents applied in human and veterinary medicine against bacterial pathogens.