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Background. Early definitive identification of infectious pathogens coupled with antimicrobial stewardship interventions allow for targeted and timely administration of antimicrobials. We investigated the combined impact of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) technology and an antimicrobial stewardship program (ASP) in pediatric patients with blood stream infections (BSIs). Methods. This is a single-center study comparing a control group of patients from October 2009 to July 2010 with BSIs to a cohort of patients postimplementation of MALDI-TOF and an ASP, from October 2013 to July 2014. Primary outcome was time to optimal therapy. Secondary outcomes included time to effective therapy, 30-day all-cause mortality, 30-day readmission rate, hospital length of stay, and intensive care admission. Results. One hundred episodes of BSIs were identified in the preintervention period, and 121 episodes were identified in the postintervention period. Time from blood culture collection to organism identification was significantly reduced in the prospective cohort compared with historical controls (18.8 vs 43.7 hours, respectively). A total of 73 ASP interventions were made on the treatment of BSIs in the postintervention period. Combined use of MALDI-TOF and ASP significantly reduced time to optimal therapy (77.0 to 54.2 hours, P < .001). In the subgroup analysis of Gram-negative bacteremia, time to effective and optimal therapy were significantly reduced (2.0 vs 0.7 hours and 146.8 vs 48.0 hours, respectively). There were no significant differences in clinical outcomes. Conclusions. The combined use of MALDI-TOF and ASP allows early optimization of antimicrobial therapy in pediatric inpatients with BSIs.