Introduction and Objectives: Knowledge of hand hygiene is important for medical students. The aim of this study was to compare the knowledge before and after a workshop on hand hygiene held for medical students at the Faculty of Medicine, General Sir John Kotelawala Defence University, Sri Lanka. Methods: A self-administered, pre-tested validated questionnaire, based on hand hygiene guidelines of the World Health Organization (WHO), was distributed among the medical students before and after conducting a workshop on hand hygiene.Results were assessed by comparing the current guidelines set by the WHO with the knowledge of hand hygiene among the medical students. Results: All 177 students participated in the study before conducting the workshop. There were 104 (58.8%) preclinical and 73 (41.2%) clinical students. Of the 104 preclinical students, the percentage who knew the importance of “My five moments for hand hygiene” approach (hand hygiene before direct contact with patients, after direct contact with patients, before clean/aseptic procedures, after contact with blood/body fluid and after contact with patient’s surrounding) before conducting the workshop were 53.8%, 67.3%, 51.0%, 88.5% and 34.6% respectively. Of the 73 clinical students, the percentage who knew the importance of the “My five moments for hand hygiene” approach before conducting the workshop were 49.3%, 63.0%, 87.7%, 94.5% and 27.4% respectively. Of the 112 students who participated in the workshop, there were 68 (60.7%) preclinical and 44 (39.3%) clinical students. Of the 68 preclinical students, 77.9%, 79.4%, 91.2%, 95.6% and 70.6% knew the importance of the “My five moments for hand hygiene” approach post workshop. Post workshop, the percentage of the 44 clinical students who knew the importance of “My five moments for hand hygiene” approach were 90.9%, 88.6%, 93.2%, 97.7% and 81.8% respectively. Conclusions: The pre workshop knowledge of hand hygiene among the two categories of medical students was not satisfactory. The knowledge on each component of “My five moments for hand hygiene” concept improved to more than 70% after conducting the workshop.