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We aim to assess the state-of-the-art of Infection Prevention and Control (IPC) practices and their results in Italian healthcare facilities 6 years after the kick-off of the new National Plan for the Control of Antibiotic Resistance (PNCAR). This was a multicenter, cross-sectional, observational study conducted between January 1st, and December 31st, 2023, among Italian hospitals. Survey questions were readapted from the WHO IPC assessment framework (IPCAF), PNCAR and INSIEME group proposals, resulting in 155 questions grouped into six sections: IPC program, surveillance, control activities for healthcare-associated infections, antimicrobial stewardship (AMS) strategies, IPC training and education, and monitoring indicators. Thirty-eight acute care Hospitals completed the survey (response rate: 30%): 26.3% of hospitals did not define an annual program and 34.2% an AMS task force. Periodic microbiology reports were available for 57.9%, hospital alcohol-based hand rubs consumption per 1000 bed-days in 42.1% and defined daily dose of antimicrobials per 100 bed-days in 55.3% hospitals with a significant difference between Northern and Southern regions (90.9% vs 22.2%; P = 0.006). Active monitoring of hand hygiene and contact isolation compliance was implemented in only half of the hospitals. Structured HAI surveillance systems were implemented in fewer than 20% of hospitals. Components on IPC implementation activity, such as no-touch sanitizing systems, checklist for environmental cleaning, informatic flags, bundles for pathogens revealed the lowest score. The appropriateness of antibiotic therapy prescribing was assessed in 73.7% of facilities. Only 42.1% of hospitals had budget goals. This survey provides a baseline assessment and identifies key barriers to the implementation of IPC programs across Italian acute care hospitals. The findings highlight priority areas for intervention and will inform the next phase of the INSIEME project, which aims to develop and implement tailored strategies that address the specific needs of healthcare workers and institutions nationwide.