Infective endocarditis (IE) is a life-threatening disease, establishing a diagnosis is often challenging. The aim of this prospective study was to evaluate and compare the diagnostic performance of the combined use of single photon emission tomography and computed tomography with technetium99m-hexamethylpropyleneamineoxime—labeled leukocytes (99mTc-HMPAO-SPECT/CT) with transthoracic echocardiography (TTE) in patients with suspected IE. We enrolled 40 consecutive patients (12 females, 28 males, mean age: 58.6 ± 18) with suspected IE in the years 2015–2016. All patients underwent clinical evaluation, TTE and 99mTc-HMPAO-SPECT/CT for the assessment of lesions typical for IE. Scans were evaluated for the presence and location of increased radioactivity foci, corresponding to the accumulation of radiolabeled leukocytes in inflammatory lesions. After 6 months, the patients were re-evaluated clinically and with TTE. Final IE diagnosis was established in 14 (35%) patients. Lesions typical for IE were shown in 28 (70%) TTEs and 16 (40%) 99mTc-HMPAO-SPECT/CTs. The latter tests were characterized by 90% accuracy, 93% sensitivity, 88% specificity, 96% negative predictive value (NPV), 81% positive predictive value (PPV). TTE demonstrated 60% accuracy, 93% sensitivity, 42% specificity, 92% NPV, and 46% PPV. 99mTc-HMPAO-SPECT/CT was characterized by a lower number of false-positive results compared to TTE (3 vs. 15). In patients with suspected IE, 99mTc-HMPAO-SPECT/CT yields a smaller number of false-positive results, significantly higher diagnostic accuracy, specificity and PPV than TTE. It helps to differentiate IE infectious and sterile echocardiographic lesions and reduces by 27% the number of misdiagnosed IE classified in the ‘possible IE’ category by modified Duke Criteria.