Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) decreases the time to organism identification and improves clinical and financial outcomes. The purpose of this study was to evaluate the impact of MALDI-TOF MS alone versus MALDI-TOF MS combined with real-time, pharmacist-driven, antimicrobial stewardship (AMS) intervention on patient outcomes. This single-center, pre-post, quasiexperimental study evaluated hospitalized patients with positive blood cultures identified via MALDI-TOF MS combined with prospective AMS intervention compared to a control cohort with MALDI-TOF MS identification without AMS intervention. AMS intervention included: real-time MALDI-TOF MS pharmacist notification and prospective AMS provider feedback. The primary outcome was the time to optimal therapy (TTOT). A total of 252 blood cultures, 126 in each group, were included in the final analysis. MALDI-TOF MS plus AMS intervention significantly reduced the overall TTOT (75.17 versus 43.06 h; P Ͻ 0.001), the Gram-positive contaminant TTOT (48.21 versus 11.75 h; P Ͻ 0.001), the Gramnegative infection (GNI) TTOT (71.83 versus 35.98 h; P Ͻ 0.001), and the overall hospital length of stay (LOS; 15.03 versus 9.02 days; P ϭ 0.021). The TTOT for Grampositive infection (GPI) was improved (64.04 versus 41.61 h; P ϭ 0.082). For GPI, the hospital LOS (14.64 versus 10.31 days; P ϭ 0.002) and length of antimicrobial therapy 24.30 versus 18.97 days; P ϭ 0.018) were reduced. For GNI, the time to microbiologic clearance (51.13 versus 34.51 h; P Ͻ 0.001), the hospital LOS (15.40 versus 7.90 days; P ϭ 0.027), and the intensive care unit LOS (5.55 versus 1.19 days; P ϭ 0.035) were reduced. To achieve optimal outcomes, rapid identification with MALDI-TOF MS combined with real-time AMS intervention is more impactful than MALDI-TOF MS alone.KEYWORDS MALDI-TOF MS, antimicrobial stewardship, rapid molecular diagnostics D espite advances in antimicrobial therapy, bloodstream infections (BSIs) remain a threat to hospitalized patients. A significant proportion of health care-associated infections result from multidrug-resistant organisms (MDROs). These infection rates continue to uptrend, posing a substantial public health risk by driving providers to utilize broad-spectrum antimicrobials and potentiating the cycle that creates MDROs