Introduction: The guidelines issued by the WHO in 2009 on hand hygiene in healthcare have provided medical professionals with scientific evidence to justify the need to comply with hand hygiene practices when treating patients. Aim of the study: The aim was to examine the doctors' and nurses' knowledge of the "five moments for hand hygiene" by the WHO. Material and methods: The study was carried out using a questionnaire devised by the authors of the study; random sampling was used. The study involved 231 doctors and nurses working in a multi-profile hospital in Lesser Poland in 2017. Results: 75.9% of respondents admitted knowing the "five moments for hand hygiene", 12.9% said they did not know them, and 10.8% abstained. Most often, respondents listed the first point (before patient contact-81.7%), followed by, respectively: after patient contact-79.1%; after body fluid exposure risk-59.2%; before aseptic task-58.1%; and after contact with patient surroundings-51.8%. Better knowledge of the "five moments for hand hygiene" was shown by women than by men, by nurses than by doctors, and by people with seniority of less than 20 years. Employees of medical treatment wards presented more knowledge in this regard than employees of surgical departments and ICUs, while the staff of the Emergency Room had the lowest knowledge. Conclusions: Some respondents did not know the guidelines concerning the "five moments for hand hygiene" by the WHO, and the level of knowledge of those who declared good knowledge of the subject was insufficient.