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Background: Healthcare-associated infections are major causes of morbidity and mortality associated with clinical, diagnostic, and therapeutic procedures in healthcare organizations. Enhancement of hand hygiene practices among healthcare workers is responsible for decreasing 30 to 50% of healthcare-associated infections. However, previous studies have indicated inadequate adherence to hand hygiene procedures in healthcare institutions. Therefore, increasing knowledge, attitude, and practices of hand hygiene among nursing students who participate in caring for patients is essential to improve the quality of care and patient safety. Objectives: This study aimed to examine knowledge, attitude, the practice of hand hygiene, and their associations among nursing students at Can Tho University of Medicine and Pharmacy. Materials and methods: A descriptive correlational study was conducted with 104 nursing students as study participants. Knowledge and attitude were examined by using a self-structured questionnaire, while practice was evaluated by observation based on the hand hygiene checklist of the Vietnamese Ministry of Health. Results: The findings showed that 44.2% and 66.3% of participants had good knowledge and positive hand hygiene attitudes, respectively. Poor adherence of 5 moments of hand hygiene and 6 steps of the hand hygiene procedure were revealed. Half of the respondents showed low to moderate compliance with the hand hygiene procedure. There were significant associations between knowledge, attitude, and hand hygiene practices among nursing students. Conclusion: among nursing students, poor knowledge, attitude, and hand hygiene practices were reported. The study findings supported previous literature and found that respondents emphasized the importance of training programs and hand hygiene monitoring in improving hand hygiene practices.