Objective: The purpose of this study is to assess the awareness and knowledge levels of the students, trainees and faculty members of a university hospital on hand hygiene. Methods: This study was conducted with a total of 1266 people, 892 students, 243 trainees and 131 faculty members studying or working at a university hospital. The questionnaires was prepared after a literature search, and was administered to the participants through face-to-face interviews. Results: Out of 1266 participants, 657 (51.9%) were male. Among them, 91% of the students and 73.2% of the trainees were found to have received hand hygiene training within the last 5 years; and 23.1% of the trainees and 38.7% of the students had received such training within the last year. None of the faculty members had received hand hygiene training within the last 5 years. Those who received training within the last year had the highest knowledge level (p=0.001). The knowledge level of the students was found higher than those of trainees and faculty members (p=0.01). Compliance with hand hygiene was rated moderate in 54.2% of the participants, good in 23.1% and poor in 32.7%. The information known correctly by most of the participants was “gloves should be replaced in every patient” and the information known incorrectly by the majority was “wearing gloves is a substitute for hand hygiene”. The major reason for noncompliance with hand hygiene was high workload. Conclusions: We found the importance of ensuring continuation of the efforts towards increasing compliance with hand hygiene in our hospital and that trainings should cover all the staff working at the hospital (particularly the physicians).