The multicenter ENDOBACT study aimed at implementing molecular methods for identification of bacterial species encountered in infective endocarditis, and at attempting to reduce the number of cases with unknown etiology. For eighty seven cases was established a diagnosis of definite infective endocarditis. Thirty two of these cases had negative blood cultures. For nine cases out of 32, valve pieces were available and an attempt was made to identify the etiological agent by molecular techniques. Thirty seven available isolates were identified by phenotypical and molecular comparative methods: 16S rRNA (all available isolates), rpoB (staphylococci, streptococci and enterococci), sodA (streptococci and enterococci) genes sequencing. For eight isolates, the comparative results were discrepant. Species identification of one coagulase negative staphylococcal strain was assigned using molecular methods. Molecular identification methods applied here might represent an added value for clinical and conventional microbiological diagnosis of infective endocarditis in Romania.