Aims and objectives: To evaluate the knowledge, attitude and practice on hand hygiene among registered nurses and nursing students in a referral hospital. Background of study: HCAIs is associated with increased morbidity, mortality, prolonged hospital stay, increased health care costs, and antibiotic resistance in both acute and long term care facilities. Based on recommendations from WHO and the Centers for Disease Control, HH is the most important and easy way for the control of hospital infections. The five moments of hand hygiene opportunities by WHO is a simple schema which can be remembered and put into practice by prudent nurses. Design: A quantitative survey approach was used to collect data from the registered nurses and students of a referral hospital. Results: The mean knowledge of entire sample (registered nurses and students) on WHO hand hygiene questionnaire in our study was 13.9 (moderate knowledge) and the percentage was 60.8. Moreover there was no significant difference among the registered nurses and students in their knowledge level. Furthermore a positive attitude and practice was observed among registered nurses and students. Conclusion: The study highlights the importance of regular services of in-service education for registered nurses which can help to prevent health care associated infections to a great extent. The nurse educators and practitioners should serve as role models and emphasize the importance of hand hygiene to budding as well as registered nurses for improved patient care outcomes.