The ability of enterococci to acquire resistance to antibiotics and form biofilms in vivo makes these infections, endocarditis in particular, especially difficult to treat. A collection of clinical enterococcal isolates was screened for the presence of various virulence determinants and in an in vitro assay for biofilm formation. Isolates were chosen for the presence or absence of the genes for Esp and gelatinase and different in vitro biofilm phenotypes, and were evaluated in a rat model of endocarditis; all colonized vegetations to similar degrees. Treatment with vancomycin resulted in a 2.7-log reduction in colony-forming unit (CFU) in vegetations for an esp(+)/gel(-) strain, compared with no reduction in CFU for an esp(+)/gel(+) or an esp(-)/gel(-) isolate. These results suggest that although there may not be an absolute role for individual virulence determinants in infectivity, combinations of factors may play a role in allowing a biofilm infection to be more resistant to therapy.