Background: Healthcare-associated infection (HCAI) is defined as any infection that the patient gets during hospitalization or after discharge, which was not present at the time of admission. Several factors contribute to HCAIs such as inappropriate hand hygiene, which is considered the most important and simplest precautionary measure to reduce the prevalence and control infection. This study aimed to assess the knowledge, attitude, and practices of hand hygiene among clinical year medical students of Imam Mohammad Ibn Saud Islamic University (IMSIU), Riyadh, Saudi Arabia. Methods: A cross-sectional survey of 240 clinical year medical students (4th and 5th year) at IMSIU was conducted using a validated self-administered questionnaire. It was cross-sectionally distributed to our participants. Results: Total 196 male students (n=196/240) participated in the questionnaire with a response rate of 81.7%. All participants were male students with the mean age of 23 years old. Overall, the level of knowledge, attitude and practices of hand hygiene among clinical year medical students at IMSIU, Riyadh, Saudi Arabia was found to be moderate (50%-75%). Conclusion: The present study warrants for systematic educational programs for students’ awareness regarding the knowledge, attitude and practice towards hand hygiene use and implementation of strict guidelines to regulate nosocomial infection.