Background: Methicillin-resistant Staphylococcus aureus (MRSA) remains one of the most common causes of health care-associated infection (HAI). Objective: To evaluate the effect of education and a tiered, evidence-based infection prevention strategy on rates of hospital-onset MRSA bloodstream infection (BSI). Design: Prospective, national, nonrandomized, interventional, 12-month, multiple cohort, pre-post observational quality improvement project. Setting: Acute care, long-term acute care, and critical access hospitals with a disproportionate burden of HAI. Patients: All patients admitted to participating facilities during the project period. Intervention: A multimodal infection prevention intervention consisting of recommendations and tools for prioritizing and implementing evidence-based infection prevention strategies, ondemand educational videos, Internet-based live educational presentations, and access to content experts. Measurements: Rates of hospital-onset MRSA BSI, overall and stratified by hospital type, during 12-month baseline and postintervention periods. Variation in outcomes across hospital types was examined. Results: Between November 2016 and May 2018, 387 hospitals in 23 states and the District of Columbia participated, 353 (91%) submitted MRSA data, and 172 (49%) indicated that MRSA prevention was a priority. Unadjusted overall rates of hospital-onset MRSA BSI were 0.075 (95% CI, 0.065 to 0.085) and 0.071 (CI, 0.063 to 0.080) per 1000 patient-days in the baseline and postintervention periods, respectively. Limitations: The intervention period was short. Participation and adherence to recommended interventions were not fully assessed. Baseline rates of hospital-onset MRSA BSI were relatively low. Prevention of MRSA was a priority in a minority of participating hospitals. Patient characteristics and other MRSA risk factors were not assessed. Conclusion: In hospitals with a disproportionate burden of HAIs, access to tools to assist with implementation of evidencebased prevention strategies and education resources alone may not be sufficient to prevent MRSA BSI.