The need to investigate novel dosing regimens and combinations is essential in combating poor treatment outcomes for Staphylococcus aureus bacteremia and endocarditis. We evaluated the impact of simulated standard- and high-dose daptomycin in combination with gentamicin or rifampin against daptomycin-susceptible and nonsusceptible matched strains of S. aureus. These strains were collected from the daptomycin bacteremia and endocarditis clinical trial and consisted of three susceptible strains (MIC, 0.25 mg/liter) and four nonsusceptible isolates (MICs, 2 to 4 mg/liter). Daptomycin regimens of 6 and 10 mg/kg of body weight daily alone and in combination with gentamicin at 5 mg/kg daily or rifampin at 300 mg every 8 h were evaluated using an in vitro model with simulated endocardial vegetations over 96 h. Rapid bactericidal activity, identified by time to 99.9% kill, was displayed in all regimens with the daptomycin-susceptible strains. Concentration-dependent activity was noted by more-rapid killing with the 10-mg/kg/day dose. The addition of gentamicin improved activity in the majority of susceptible isolates. Daptomycin 6-mg/kg/day monotherapy displayed bactericidal activity for only one of the nonsusceptible isolates and for only two isolates with increased doses of 10 mg/kg/day. Combination regimens demonstrated improvement with some but not all nonsusceptible isolates. Three isolates developed a reduction in daptomycin susceptibility with 6-mg/kg/day monotherapy, but this was suppressed with both combination therapy and high-dose daptomycin. These results suggest that high-dose daptomycin therapy and combination therapy may be reasonable treatment options for susceptible isolates; however, more investigations are needed to confirm the variability of these regimens with nonsusceptible isolates.