Healthcare associated infections (HCAI) are huge problem all over the world, and 5-10% of all hospitalized patients will develop infection during hospitalization. From the times of I. P. Semelweiss we know that clean hands are the most important single factor that can decrease the number of HCAI. World Health Organization (WHO) has recognised this problem and developed Guidelines for hand hygiene in healthcare institutions. This also was the reason of developing Croatian national Guidelines. The main goal of the Guidelines was to decrease number of HCAI associated with the hands of healthcare workers. These Guidelines are meant for all healthcare workers and other hospital staff who come to the direct contact with patients. An interdisciplinary team of experts developed these Guidelines using WHO Guidelines, other existing guidelines and literature reviews for hand hygiene. Grades of evidence for specific recommendations were determined using CDC/HICPAC grading system. Categorization is based on existing data, theoretical basis, applicability and economic impact. After a broad discussion in different professional societies, Guidelines were accepted. Guidelines include recommendations for hand hygiene indications, hand hygiene technique, surgical hand preparation, choosing hand hygiene preparations, skin care, nails, glove use, patients and visitors hand hygiene, role of education, as well as role of healthcare institution and role of government. Furthermore, in the Guidelines the concept of "Five moments for hand hygiene" is explained in detail, and main literature data are presented.