Abstract Background Healthcare-associated infections cause a significant burden of disease globally. Hand hygiene is crucial for preventing these infections but despite recommendations, compliance with hand hygiene protocols among healthcare professionals is often inadequate. In Portugal, there have been several initiatives aiming to increase awareness on the importance of hand hygiene practices amongst healthcare professionals including in primary healthcare facilities. Nationally, there are two models of organization of primary healthcare facilities with differences in funding and allocation of resources. Methods In this study, the World Health Organization’s Hand Hygiene Knowledge Assessment Questionnaire for Healthcare Professionals was translated into Portuguese and validated with infection control specialists. It was then distributed to primary healthcare professionals in the Northern Lisbon area, in March 2024 to assess knowledge on hand hygiene practices and highlight potential training needs. The responses were analyzed, with the Kruskal-Wallis test carried out, followed by the Mann-Whitney U test when required. Results A response rate of 21,5% (65/302) was obtained, with most participants being nurses (58%), followed by physicians (34%). There was a statistically significant difference depending on the model of organization of the primary healthcare unit to which the participant belonged (χ2 = 8.428, p = 0.014, df = 2)., There was no significant difference between the score and the professional category of the participant (χ2 = 5.76, p = 0.12, df = 3). Conclusions The findings of this study show that the model of organization of a primary healthcare unit can impact the knowledge of professionals and not just practices. This study also underscores the significance of comprehensive assessments of the context before interventions and the need for further studies to understand how we can better inform future training initiatives. Key messages • Organizational differences in primary healthcare units impact hand hygiene knowledge among professionals, emphasizing the need for tailored training initiatives. • Comprehensive assessments of healthcare contexts, coupled with validated linguistic tools, are crucial for reliable intervention strategies in infection control.