Introduction: Hand hygiene plays a significant role in controlling the spread of health-care infections. As nurses are in direct contact with patients for a significant proportion of the time, they can be a vehicle for cross-contamination within the hospital and as such, good hand hygiene protocols can ensure the safety of patients in a variety of settings. The aim of this study is to assess the knowledge and perception of hand hygiene among nurses in Saudi Arabian hospitals. Materials and Methods: A cross-sectional study was conducted between November 2017 and May 2018 to determine the nurses' knowledge of hand hygiene. Nurses from six hospitals in the Asir region of Saudi Arabia were recruited using multi-stage cluster sampling. The World Health Organization Knowledge and Perception Questionnaire for Health-care Workers were administered to collect the information. Results: During the recruitment phase, 300 nurses were contacted and 243 agreed to participate in the study, indicating a response rate of 81%. About 51.85% of the nurses demonstrated a good level of knowledge about hand hygiene and 50.2% of nurses had a negative perception of hand hygiene. The results have shown a positive association between knowledge, nurses' perceptions, and years of experience, and their participation on a training course about hand washing. Conclusion: Educational interventions should be undertaken to enhance the knowledge of nurses/health-care providers and to promote positive perceptions of hand hygiene among nurses.