Background:Hand hygiene (HH) is considered the most beneficial method to reduce the incidence of Hospital-Acquired Infections. However, compliance to HH by healthcare workers are reportedly poor with a compliance rate of 40%. In spite of the reports concerning the knowledge of HH among medical students in many countries, Saudi Arabia has greatly lagged behind in this aspect. Aim: This study aims to evaluate HH knowledge and practice among first and last clinical years; (i.e. fourth and sixth year respectively) at King Abdulaziz University Hospital (KAUH), Jeddah, Saudi Arabia. Methods: A cross-sectional study was conducted on 261 medical students of fourth and sixth year, at KAUH in Jeddah, Saudi Arabia. A covert observation was used to assess the practice and a 5-point Likert scale questionnaire was used for evaluating the students' knowledge. Both were based on the WHO five moments of HH. A consent was obtained from all participants. Data analysis was done using SPSS version 21 and Pearson's Chi-Square was used for association. Results: Medical students included in the study were fourth and sixth year students accounting as 157 (60%), and 104 (40%) respectively. Practice among sixth year students was found to be better than fourth year students (p = 0.004). As for the knowledge between fourth year and sixth year students it was found to be similar and markedly high (p = 0.5), ranging between strongly agree and agree in the questionnaire. Conclusion: Strict protocols are needed for the implementation of HH practice among students.