Background: Acute pulmonary thromboembolism (APTE) is a life-threatening condition, often manifesting with chest pain, dyspnea, and increased cardiac biomarkers including cardiac troponin I (CTI) and D-dimer. Therefore, APTE is often misdiagnosed with classical non-ST elevation myocardial infarction (NSTEMI), resulting in unnecessary coronary interventions and a delay of therapy. Objectives: Our aim was to distinguish APTE from NSTEMI based on CTI and D-dimer levels. Methods: Complete clinical and laboratory data sets from APTE patients (n = 123) were compared with matched NSTEMI patients (n = 123) who presented with chest pain. The APTE diagnosis was confirmed by chest tomography, angiography, or radionuclide ventilation-perfusion scan, while NSTEMI was established by clinical symptoms, cardiac biomarkers, and coronary angiography. Clinical characteristics, CTI (initial and peak), and D-dimer levels at presentation were retrospectively analyzed. Results: The clinical characteristics were not different between APTE and NSTEMI patients. However, significantly lower initial CTI (0.2 ± 0.5 vs. 4.4 ± 9.5 ng/ml) and peak CTI (0.7 ± 2.7 vs. 17.1 ± 20.4 ng/ml), but higher initial D-dimer (9.8 ± 9.4 vs. 1.6 ± 3.6 ng/ml), distinguished APTE from NSTEMI. By receiver operating characteristic curve analysis, the cutoff values for initial CTI, peak CTI, and D-dimer were 0.25, 0.98, and 3.18 ng/ml, respectively. Conclusion: Patients with APTE exhibited lower initial and peak CTI but higher D-dimer levels than NSTEMI patients. Assessing cardiac biomarkers is useful for differentiating APTE from NSTEMI. Further large randomized biomarker studies are urgently needed to facilitate a better APTE diagnosis since clinical characteristics are not particularly helpful.