The aim of this study was to determine the diagnostic value of F-fluorodeoxyglucose (FDG) positron emission tomography and computed tomography (PET/CT) and magnetic resonance imaging (MRI) in diagnosing vertebral osteomyelitis. From November 2015 until December 2016, 32 patients with suspected vertebral osteomyelitis were prospectively included. All patients underwent both F-FDG-PET/CT and MRI within 48 h. All images were independently reevaluated by two radiologists and two nuclear medicine physicians who were blinded to each others' image interpretation. F-FDG-PET/CT and MRI were compared to the clinical diagnosis according to international guidelines. For F-FDG-PET/CT, sensitivity, specificity, PPV, and NPV in diagnosing vertebral osteomyelitis were 100%, 83.3%, 90.9%, and 100%, respectively. For MRI, sensitivity, specificity, PPV, and NPV were 100%, 91.7%, 95.2%, and 100%, respectively. MRI detected more epidural/spinal abscesses. An important advantage of F-FDG-PET/CT is the detection of metastatic infection (16 patients, 50.0%). F-FDG-PET/CT and MRI are both necessary techniques in diagnosing vertebral osteomyelitis. An important advantage of F-FDG-PET/CT is the visualization of metastatic infection, especially in patients with bacteremia. MRI is more sensitive in detection of small epidural abscesses.