Abstract Background Healthcare-associated infections (HAIs) are the main complications of hospitalization. The goal of the study was to enhance awareness, improve risk perception, and facilitate measures to reduce the outcomes of HAIs. A bottom-up approach, in which the healthcare workers involved play a key role, can be adopted to decrease the spread of HAIs. Therefore, a survey was conducted in a large teaching hospital in Rome from 2019 to 2023, excluding 2020 due to COVID-19. Methods A longitudinal study was conducted in all integrated care departments with beds. The data was collected using a standardized, anonymous, self-administered questionnaire for healthcare workers. The questionnaire consisted of 61 items assessing socio-professional features, knowledge of HAIs, attitudes and barriers to hygiene compliance, self-analysis of professional behavior and advice for new interventions. The study lasted 21 days between November and December, which is considered a period of normal care activity. A multivariable logistic regression model was built. Statistical analyses were performed with STATA 18.0. Results There were 384 health workers respondents: female (67.7%) and nurses (56.5%). The most represented areas were internal medicine (37.9%) and surgery (27.3%). The correct frequency of perception of HAIs increased from 33.3% in 2019 to 36.3% in 2023. From 2022 to 2023, the multivariable model showed that physicians (OR 0.48, CI 95% 0.24-0.93, p-value 0.03) and elder staff (OR 1.02, CI 95%, 1.0-1.1, p-value 0.04) seemed to be more aware of the prevalence of HAIs in their ward. Conclusions Targeted training days were organized after the study to discuss the data collected. Staff awareness of the prevalence of HAIs in their ward is improving, even if it is complicated to quantify it by attending HAIs training events. The survey has been included as a corporate goal for 2024 as a useful tool to improve awareness of HAIs and to evaluate the effectiveness of ongoing training. Key messages • A bottom-up approach, in which healthcare workers assume a pivotal role, can be employed to reduce the spread of HAIs as it serves as a valuable tool to evaluate the efficacy of targeted training. • The results indicate that the education of healthcare workers is a crucial factor in enhancing awareness of HAIs. Therefore, there is a need to improve the education of younger and non-medical staff.