Management of central venous catheters (CVC) is a frequent procedure in intensive care units (ICUs) and the risk of bloodstream infections (CLABSI) is found to be high. The literature provides healthcare professionals with guidelines to prevent the risk of CLABSI infections. The aim of this study was to observe the knowledge, attitudes, and practices of ICU nurses on the prevention of CLABSI. A multicenter cross-sectional study was conducted between March 2023 and September 2023; the research was carried out among six ICUs in Bari both on the web and in paper mode. The survey consisted of multiple-choice questions structured in two sections: demographic sample data and the 2017 Esposito MR questionnaire, drawn up based on the 2011 guidelines of the Centers for Disease Control and Prevention, consisting of four dimensions: knowledge, attitudes, practices, information. 121 nurses (57.1%) participated in the study. 72% were unfamiliar with the guidelines, especially nurses aged 41-50 years (RR = 1.88; CI = 0.78-4.51; p = 0.13) and more than 10 years' work experience (RR = 1.56; CI = 0.76-3.23; p = 0.20). Regarding attitudes, nurses were aware of the usefulness of the guidelines (Me = 10; IQR = 8-10) and the importance of hand washing for prevention (Me = 10; IQR = 10-10) despite 39.7% (n = 48) believing that gloves replace hand washing, and there were no statistically significant differences in comparing scores with age, work experience, and educational qualification. 96.7% always substituted dressing for CVC; 120 nurses (99.2%) were always replacing infusion sets and 71.2% always sanitized the access ports before infusions. Finally, 102 participants (84.3%) felt the need to get more information about the prevention of CLABSI. We observed a reduced knowledge of the guidelines for the prevention of CLABSI despite the attitudes and practices of the nurses being satisfactory. In addition to implementing training, strategies for disseminating guidelines should be adopted.