Background: Hand hygiene (HH) is the simplest and the most fundamental means of hospital-acquired infection (HAI) prevention in both hospitals and long-term care facilities (LTCFs) which differ as to their structure, organization and epidemiology. The objective of this study was to evaluate the knowledge of, and attitudes towards, compliance with the HH guidelines by medical staff of LTCFs and hospitals, in the context of infection control organization. Material and Methods: The study was carried out among medical staff of LTCFs and hospitals using an anonymous questionnaire designed by the authors. The questionnaire was composed of 22 questions. Results: Among 237 healthcare workers from LTCFs and hospitals (51.5% vs. 48.5%), the vast majority were women (97.5% vs. 94.8%), who were nurses (86.9% vs. 91.3%) with 21–30 years of experience (28.5% vs. 44.3%). The respondents, both working in hospitals and in LTCFs, declared that there was some surveillance of HAIs in their workplace – 78.8% vs. 87.8%, respectively, p = 0.082. However, the respondents from LTCFs significantly more often than those working in hospitals declared the lack of HAI registration (12.3% vs. 0.9%, p = 0.002), as well as the lack of surveillance of multidrug-resistant microorganisms (16.4% vs. 4.3%, p = 0.010). Although the knowledge of WHO HH guidelines was declared by over 90% of the respondents, only about 70% of them (with no significant difference between both types of facilities) properly indicated the 5 moments of HH. Conclusions: The results of the study indicate that the organizational conditions and practice of HH in LTCFs and hospitals present some differences. Therefore, there is a need for observational studies concerning HH in the context of the structure and organization of infection control, as they are necessary for the development and implementation of effective programs to improve the situation in this field. Med Pr. 2020;71(5):531–7