The aim of the study was to investigate the value of F-fluorodeoxyglucose positron-emission tomography combined with computed tomography (F-FDG-PET/CT) in diagnosing native valve endocarditis (NVE). All patients with bacteremia and suspicion of NVE between January 2013 and June 2016 were identified from the hospitals' register and retrospectively included if echocardiography and F-FDG-PET/CT were performed within 14 days. F-FDG-PET/CT scans were scored independently by two nuclear medicine physicians. F-FDG-PET/CT was compared with the modified-Duke criteria and a multidisciplinary consensus. A total of 88 patients were included. In 10 patients with definite NVE according to the modified-Duke criteria, three (30.0%) patients had increased F-FDG uptake in or around the heart valves and seven (70.0%) patients had no increased F-FDG uptake. In patients without definite NVE according to the modified-Duke criteria, 89.7% (70/78) of the patients had no increased F-FDG uptake in or around the heart valves. Of all 20 patients with NVE according to multidisciplinary consensus, nine (45.0%) patients had increased F-FDG uptake in or around the heart valves and 11 (55.0%) patients had a normal F-FDG-PET/CT result. A negative F-FDG-PET/CT result should not be interpreted as an exclusion of NVE. In patients with possible or rejected NVE according to the modified-Duke criteria, F-FDG-PET/CT could be used in case of sustained suspicion of NVE owing to its high specificity in case of abnormal FDG uptake at the valve region. F-FDG-PET/CT is important for detecting metastatic infection which already warrants the need to perform F-FDG-PET/CT in all patients with suspected NVE.