Objective: To assist hospitals in reducing Clostridioides difficile infections (CDI), the Centers for Disease Control and Prevention (CDC) implemented a collaborative using the CDC CDI prevention strategies and the Targeted Assessment for Prevention (TAP) Strategy as foundational frameworks. Setting: Acute-care hospitals. Methods: We invited 400 hospitals with the highest cumulative attributable differences (CADs) to the 12-month collaborative, with monthly webinars, coaching calls, and deployment of the CDC CDI TAP facility assessments. Infection prevention barriers, gaps identified, and interventions implemented were qualitatively coded by categorizing them to respective CDI prevention strategies. Standardized infection ratios (SIRs) were reviewed to measure outcomes. Results: Overall, 76 hospitals participated, most often reporting CDI testing as their greatest barrier to achieving reduction (61%). In total, 5,673 TAP assessments were collected across 46 (61%) hospitals. Most hospitals (98%) identified at least 1 gap related to testing and at least 1 gap related to infrastructure to support prevention. Among 14 follow-up hospitals, 64% implemented interventions related to infrastructure to support prevention (eg, establishing champions, reviewing individual CDIs) and 86% implemented testing interventions (eg, 2-step testing, testing algorithms). The SIR decrease between the pre-collaborative and post-collaborative periods was significant among participants (16.7%; P < .001) but less than that among nonparticipants (25.1%; P < .001). Conclusions: This article describes gaps identified and interventions implemented during a comprehensive CDI prevention collaborative in targeted hospitals, highlighting potential future areas of focus for CDI prevention efforts as well as reported challenges and barriers to prevention of one of the most common healthcare-associated infections affecting hospitals and patients nationwide.