The purpose of this study was to evaluate the impact of real-time PCR reporting both on timely identification of clustered Grampositive cocci (GPC) in blood cultures and on appropriate antibiotic treatment. This retrospective, interventional cohort study evaluated inpatients with blood cultures positive for GPC in the pre-PCR (15 January 2009 to 14 January 2010) and post-PCR (15 January 2010 to 14 January 2011) periods. Post-PCR implementation, laboratory services completed batched PCR; results other than methicillin-resistant Staphylococcus aureus (MRSA) were reported in the electronic medical record without additional interventions. The assay's sensitivity and specificity, time to identification of staphylococcal bacteremia, and clinically relevant outcomes, including time to optimal antibiotic therapy, were evaluated. Demographic information was also collected and analyzed. Sixty-eight and 58 patients with Staphylococcus aureus bacteremia from the pre-and post-PCR periods, respectively, met inclusion criteria. Similar numbers of consecutive patients with coagulase-negative staphylococci were analyzed for comparison. The time to identification was significantly reduced post-PCR implementation (mean, 13.2 h; 95% confidence interval [95% CI], 10.5 to 15.9 h; P < 0.0001). However, the time to optimal antibiotic therapy was not significantly reduced. We conclude that implementation of a PCR assay demonstrated the potential to improve appropriate antibiotic use based on clinically meaningful and statistically significant reductions in the time to microbiologic identification. However, in order to realize this potential benefit, processes must be optimized and additional interventions initiated to facilitate providers' use of the PCR result.