Background: The aim of this study was to investigate whether the diagnosis of infective endocarditis (IE) could be improved using molecular tools in addition to standard microscopy and cultivation methods. Methods: Cultivation was performed on blood or tissue samples as recommended in the modified Duke criteria. The molecular tools included a broad-range polymerase chain reaction (PCR)-based denaturant gradient gel electrophoresis and a more detailed identification by constructing clone libraries followed by sequencing. Results: Of 14 patients, 12 were positive by blood or tissue cultivation and all were monomicrobial. Molecular methods showed the presence of DNA from multiple bacterial species in 6 of the samples and indicated a larger variety of bacteria in the different samples than identified by cultivation. For 8 of the patients there was a good correlation between the results of cultivation and molecular methods, and for these samples the identified bacteria are known to be frequently involved with IE. Many of the additional bacteria only identified by the molecular methods are not reported as common causes of IE. Conclusions: Application of molecular tools in addition to cultivation indicated that polymicrobial infections might be of importance in IE. However, the significance of the more unknown microorganisms needs to be investigated further.