The management of acute chest pain is one of the challenges for emergency departments (EDs) worldwide. This study aims to provide insights into clinical and organizational aspects related to the use of different cardiac troponin tests for the diagnosis of NSTEMI. A prospective observational study was conducted among 12 Italian EDs. Eligible participants had chest pain of suspected cardiac origin and accessed EDs from January 2017 to March 2019. A 30-day follow-up was performed to gather information about the main cardiac outcomes. Tests validity and performance were assessed by computing sensitivity, specificity, positive and negative predictive values and area under the ROC curve. The independent association between adverse event end point at 30 days and type of troponin was evaluated by multiple logistic regression models, using odds ratio and 95% confidence interval. 2913 patients were included. Almost 72% were affected by comorbidities and most of them stayed in the EDs for more than 3 h, with significant differences among the different troponin assays. The results of follow-up at 30 days for the outcomes considered for the patients who were ruled out in 3 h or less did not differ significantly compared to those ruled out after 3 h or more. After adjustment for confounders, patients admitted to an ED that used a high-sensitivity troponin were at a lower risk of having a MACE (OR = 0.53, 95%CI 0.35-0.90) and a non-significant lower risk of myocardial infarction (OR = 0.68, 95% CI 0.41-1.13, p = 0.1314) at 30 days compared to patients admitted to an ED that used a standard troponin. Appropriate troponin testing is extremely important for differential diagnosis and for addressing proper treatment and safe procedures for patients who are not admitted to the hospital.