Background: Hand hygiene (HH) is the most important element of infection prevention. The aim of the study was to analyze the level of HH knowledge among medical students of Jagiellonian University Medical College in correlation with their clinical experience and the presence and extent of trainings in hospital hygiene prior to internships, as well as with HH practice among medical staff perceived by students. Material and Methods: The study was carried out in a group of 414 students from October to December, 2014. The questionnaire built of 14 questions was used as a study tool. Results: Absolutely correct answers to questions about HH were given by 52.9%, and about HH technique by 6.5% of respondents. The degree of accuracy of answers to questions concerning HH did not correlate with the gender of the respondents or with the fact that work placement had been preceded by training in the field of HH or with its scope. A statistically significant correlation was found between the year, the field, and the type of the study. Students with greater professional practice, significantly less often claimed that medical workers comply with HH. Professional practice of 22.9% of students was not preceded by any training in the field of hospital hygiene and in 28% of cases training did not cover HH. Nearly half of the respondents declared that pre-internship training had not addressed the problem of occupational exposure to biological agents. Conclusions: The results of the study shows that knowledge gained by students participating in the study was not satisfactory. Moreover, there is a need for improving the educational scheme in the discussed subject at all levels of basic and clinical subjects as well as during internships. Med Pr 2016;67(5):623–633