Background: Risk stratification of emergency department patients with suspected acute infections and/or suspected sepsis remains challenging. We prospectively validated a 29–messenger RNA host response classifier for predicting severity in these patients. Methods: We enrolled adults presenting with suspected acute infections and at least one vital sign abnormality to six emergency departments in Greece. Twenty-nine target host RNAs were quantified on NanoString nCounter and analyzed with the Inflammatix Severity 2 (IMX-SEV-2) classifier to determine risk scores as low, moderate, and high severity. Performance of IMX-SEV-2 for prediction of 28-day mortality was compared with that of lactate, procalcitonin, and quick sequential organ failure assessment (qSOFA). Results: A total of 397 individuals were enrolled; 38 individuals (9.6%) died within 28 days. Inflammatix Severity 2 classifier predicted 28-day mortality with an area under the receiver operator characteristics curve of 0.82 (95% confidence interval [CI], 0.74–0.90) compared with lactate, 0.66 (95% CI, 0.54–0.77); procalcitonin, 0.67 (95% CI, 0.57–0.78); and qSOFA, 0.81 (95% CI, 0.72–0.89). Combining qSOFA with IMX-SEV-2 improved prognostic accuracy from 0.81 to 0.89 (95% CI, 0.82–0.96). The high-severity (rule-in) interpretation band of IMX-SEV-2 demonstrated 96.9% specificity for predicting 28-day mortality, whereas the low-severity (rule-out) band had a sensitivity of 78.9%. Similarly, IMX-SEV-2 alone accurately predicted the need for day-7 intensive care unit care and further boosted overall accuracy when combined with qSOFA. Conclusions: Inflammatix Severity 2 classifier predicted 28-day mortality and 7-day intensive care unit care with high accuracy and boosted the accuracy of clinical scores when used in combination.