Background: A mixed staphylococcal infection is a frequent opportunistic infection in immunocompromised patients and is often fatal, particularly in those with severe burns or other critical conditions. While S. aureus is typically the main culprit, S. epidermidis can also contribute significantly. Minocycline, a semisynthetic tetracycline antibiotic, can be effective on its own against multi-resistant staphylococcal infections. However, its exact effectiveness against different staphylococcal species is not fully known. Aim: We, herein, studied the efficacy of minocycline against two staphylococcal species ( S. aureus ATCC-25923 and S. epidermidis ATCC-12228 ), either alone or together, by using a new in vitro pharmacokinetics / pharmacodynamics (PK/PD) system. Methodology: This PK/PD system simulates minocycline human plasma levels with a C _max of 2 and 4 mg/L, and allowed us to monitor bacterial growth spectrophotometrically based on the relative optical density (ROD) at 600 nm. Results: These two staphylococcal isolates ( S. aureus ATCC-25923 and S. epidermidis ATCC-12228 ) had minimum inhibitory concentrations (MICs) of 0.125 and 0.5 mg/L, respectively, which were tested in duplicate in an in vitro PK system with a fAUC _0-24 and a half-life of 12 h. The area under the bacterial growth curve (AUG _ROD ) was used as the PD parameter. The in vitro PK/PD relationship was then used in order to assess the efficacy of minocycline through the fAUC _0-24 -AUG _ROD relationship, which followed a sigmoid pattern (R ² =0.874). Conclusion: The in vitro PK/PD modelling of minocycline’s activity showed that the standard dose of 4 mg/L can result in a drug exposure associated with maximal efficacy against each isolate, alone or together. However, a mixed staphylococcal infection with both isolates was associated with the best minocycline efficacy compared to S. aureus alone, although it was still non-significant.