Abstract Background: Cardiac troponin is the preferred biomarker for detecting acute myocardial injury and infarction (MI). We studied whether multiple biomarkers of numerous pathophysiological pathways would increase the diagnostic accuracy for detecting MI. Methods: Seven biomarkers [myeloperoxidase, soluble CD40 ligand, placental growth factor, matrix metalloproteinase 9 (MMP-9), high-sensitivity C-reactive protein (hsCRP), cardiac troponin I (cTnI), N-terminal pro–B-type natriuretic peptide] and estimated glomerular filtration rate were measured in 457 patients presenting on admission with symptoms suggestive of acute coronary syndrome. Twenty-five patients (5.4%) received MI diagnoses. Clinical sensitivities and specificities were evaluated from 99th-percentile reference values. Forward and backward stepwise logistic regression modeling techniques were used to identify biomarkers that were independently predictive of MI. Results: Biomarker sensitivities ranged from 20% to 96%, and specificities ranged from 19% to 89%. MMP-9 had the highest sensitivity, but its specificity was 19%. cTnI demonstrated a sensitivity of 72% (95% CI, 51%–88%) and a specificity of 89% (95% CI, 85%–92%). In multivariate models, cTnI (P < 0.001) and either hsCRP (P = 0.009) or MMP-9 (P = 0.03) were independently predictive of MI. Addition of hsCRP or MMP-9 increased the specificity to 95% (95% CI, 92%–97%) or 91% (95% CI, 88%–94%), respectively, but reduced the sensitivity to 56% (95% CI, 35%–76%) and 68% (95% CI, 47%–85%) relative to cTnI alone. Conclusions: Our findings indicate that the most clinically accurate biomarker for the early diagnosis of MI is the use of cTnI alone, rather than a multiple-biomarker approach, when an analytically robust cardiac troponin assay based on the 99th percentile is used.