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Aim: To determine the relationship between knowledge, attitude and the practice of hand hygiene by nursing and midwifery students in Cambodia. Background: Hand hygiene is the most cost‐effective means to prevent hospital‐acquired infections. Techniques of hand hygiene are simple; however, many researchers have found hand hygiene knowledge, attitude and practice to be poor in many healthcare settings worldwide, especially in developing countries. Cambodia is a developing country in Southeast Asia and data regarding hand hygiene are limited. Methods: A cross‐sectional descriptive study was conducted to assess the level of knowledge, attitude and practice of hand hygiene. Students in nursing and midwifery programmes were the target population (n = 300). Survey data were collected from January to May 2017. Descriptive statistics, t‐tests and correlation coefficients were calculated to assess relationships between student knowledge, attitude and hand hygiene practice. Result: The level of knowledge, attitude and practice of hand hygiene in nursing and midwifery students was moderate. A majority of students had received hand hygiene training. There was no significant difference between nursing and midwifery students in knowledge, attitude and practice of hand hygiene. However, a Pearson correlation of attitude and practice had a weak positive relationship. Conclusion: Both nursing and midwifery students demonstrated moderate levels of knowledge, attitudes and practice of hand hygiene. Implication for nursing and health policy: This study identified a need for hand hygiene training. Developing training programmes to improve attitudes about hand hygiene is strongly recommended. Effective training may contribute to change behaviours of hand hygiene (attitude), improve practice and ultimately reduce hospital‐acquired infections. [ABSTRACT FROM AUTHOR]