Abstract Background Antimicrobial resistance (AMR) is one of the most significant public health challenges globally. The misuse or incorrect use of antibiotics has led to the emergence of resistant bacteria, making it increasingly difficult to treat infections. This study aims to identify knowledge, attitudes, and practices among Italian healthcare workers (HWs) - medical doctors (MDs) and nurses - regarding antibiotics, antibiotic use, and antibiotic resistance. Methods Cross-sectional study; a validated questionnaire from the European Centre for Disease Prevention and Control was distributed via web survey starting from April 2024 to a voluntary sample of healthcare workers employed in Italy. Descriptive analysis and multivariable logistic regression were performed to investigate predictors of knowledge regarding antibiotic use and AMR. Ethical approval was obtained from the local ethics committee. Results So far, 154 HWs were enrolled, of which 132 were nurses (85.7%). The average level of knowledge regarding AMR and antibiotic use was 6.8/7 (± 0.2) for MDs and 6.1/7 (± 0.2) for nurses, with an overall score, among HWs of 6.19 (± 0.99), compared to a European average of 6.35. The multivariable analysis highlighted that the medical profession reported higher knowledge regarding prudent antibiotic use and AMR (aOR 6.90; 95% CI 1.83-26.01, p-value = 0.004), while concerning professional practice, the unavailability and difficulty of access to guidelines seemed to increase the risk of reporting inadequate knowledge (aOR 2.04; 95% CI 1.01-4.13, p-value = 0.046). Conclusions Our preliminary results provide an insight into the importance of structuring and actively disseminating procedures/protocols and guidelines to deepen the issues of AMR and prudent antibiotic use. Also, there appears to be a need to enhance the delivery of refresher courses targeting HWs, with particular attention to settings at high infection transmission risk. EU funding: Project no. PE00000007, INF-ACT. Key messages • Although a higher knowledge about prudent antibiotic use and AMR, more efforts are needed to increase access to guidelines. • Constant updating of knowledge and practices is necessary to face the challenges of antibiotic resistance in public health.