Infective endocarditis is a rare but life-threatening disease associated with high mortality. Early diagnosis of the causative microorganism is critical to patient outcome. However, conventional diagnostic methods are often unsatisfactory in achieving this goal. As a proof of concept, we applied fluorescence in situ hybridization (FISH) for detection and identification of bacteria in histological sections of heart valves. Biopsy specimens from 54 suspected endocarditis patients were obtained during valve surgery and analysed via FISH. Specimens were screened with a probe panel that identifies the most common bacteria implicated in endocarditis. Results were compared with those of culture-based diagnostics and clinical data. Discrepant results were subjected to comparative sequence analysis of PCR-amplified 16S rRNA genes. FISH detected bacteria in 26 of the 54 heart valves. FISH allowed successful diagnosis of infective endocarditis in five of 13 blood culture-negative cases and in 11 of 37 valve culture-negative cases, showing the bacteria within their histological context. This technique allows the simultaneous detection and identification of microorganisms at the species or genus level directly from heart valves and might be a valuable tool for diagnosis of endocarditis.