Biochemical tests have a key role in the evaluation of cardiac disease. However, their overutilization and inappropriate requests create a heavy workload for laboratory and increased costs to the health care system. In 2005, we introduced new strategies for optimal use of cardiac biomarkers by practice guidelines devised by a multidisciplinary team. The adopted criteria, based on international recommendations, included information for requesting and interpreting cardiac troponin (cTn) for AMI rule-in/out and risk stratification, CK-MB mass for infarct size estimation and detection of post-PCI injury, and NT-proBNP for differential diagnosis of acute dyspnoea. One year after the implementation, a comprehensive audit was undertaken to evaluate the guideline effectiveness on test utilization and costs by comparing two one-year periods, one before (S1) and one after (S2) the protocol institution. The total test number was reduced from 201,090 to 135,017 (-33%). Myoglobin and CK-MB (as activity) were completely abolished in S2, whereas CK-MB mass showed a -87.7% reduction. No significant changes were observed for cTn (-2%), while a slight increase was demonstrated for NT-proBNP (1275 in S1 vs. 1614 in S2; +26.6%) reflecting its increasing usefulness in ED setting. The cTn workload in S2 was 14,540 tests, with 47.7% of requests coming from ED and a significant over-requesting (18.4% of total) by cardiac surgery division. Testing costs were reduced by (sic) 104,871 per annum. This audit provided valuable information on the effective running of the newly introduced cardiac biomarker protocols indicating their efficacy in reducing inappropriate biomarker requests.