We compared the pharmacodynamic activities of vancomycin and ampicillin with or without gentamicin once daily or thrice daily in an in vitro infection model with fibrin-platelet clots (FPCs) infected with Enterococcus faecalis. Antibiotics were administered as a bolus to simulate human pharmacokinetics with regimens consisting of vancomycin 1 g q12h, ampicillin 2 g q6h and gentamicin 1.3 mg/kg q8h and 5 mg/kg qd. Model experiments were performed in duplicate over 72 h. FPCs were removed from the models in triplicate at 0, 8, 24, 32, 48 and 72 h, weighed, homogenized, diluted and plated to determine colony counts. Additional FPCs were removed at over 72 h post-antibiotic dose to determine antibiotic concentrations. The inoculum density at 72 h was used to compare bactericidal activity between the regimens. Overall, all antibiotic regimens containing either ampicillin or vancomycin significantly (P < 0.01) decreased the bacterial load at 72 h compared with the growth control although monotherapy regimens with either vancomycin or gentamicin had little impact. Ampicillin was superior to vancomycin with or without the addition of gentamicin (P < 0.01). There were no significant differences in reduction of bacterial density at 72 h between the combination of ampicillin or vancomycin plus gentamicin q8h and ampicillin or vancomycin plus gentamicin once daily. This was despite achieving unmeasurable FPC gentamicin concentrations after the 8 h time point during the once-daily aminoglycoside regimen. Vancomycin plus gentamicin either every 8 h or once daily was significantly (P < 0.01) better than vancomycin alone. Ampicillin plus either of the two gentamicin regimens was also better than ampicillin alone but this did not reach statistical significance. Our data suggest that once-daily gentamicin in combination with ampicillin or vancomycin demonstrates equivalent bacterial reductions to combination therapy with thrice-daily gentamicin. Once-daily aminoglycoside combination therapy for the treatment of enterococcal endocarditis warrants further investigation.