•Characteristics of patients with infective endocarditis (IE) have changed over time.•Modified Duke criteria provide excellent diagnostic value in suspected IE.•Transesophageal echocardiography as a single diagnostic test, best detects IE.•Immunosuppression or cancer are not linked with increased IE probability. To determine whether relevant comorbidities stratify patients with and without IE and whether these may improve the diagnostic accuracy, in addition to the modified Duke criteria. 261 consecutive patients (aged 60.1 ± 16.1 years, 62.8% male) with suspected IE were prospectively included in this single-center observational trial. Modified Duke criteria and relevant comorbidities as well as clinical characteristics, were assessed. Forty-seven patients had IE, as confirmed by a clinical event committee. Patients with IE had a higher frequency of positive blood cultures (70.2% vs. 36.9%, p < 0.0001), embolic diseases (36.2% vs. 10.8%, p < 0.0001), heart murmurs (27.7% vs. 11.7%, p = 0.01), and intensive care therapy (74.5% vs. 58.4%, p = 0.04). In receiver operating characteristics, the combination of modified Duke criteria without transesophageal echocardiography led to an area under the curve of 0.783 (0.715–0.851). The predictive value was only marginally improved by the addition of heart murmur and intensive care therapy (0.794 0.724–0.863). In contrast, transesophageal echocardiography alone achieved an area under the curve of 0.956 (0.937–0.977) and was further improved when adding modified Duke criteria, heart murmur, and intensive care therapy (0.999 0.998–1.000). Modified Duke criteria provide excellent diagnostic value for evaluating suspected IE, mainly driven by transesophageal echocardiography. NCT03365193.