This study aimed to assess hand hygiene knowledge, perception, and practices of visitors to the Prophet’s Mosque in Al Madinah City, Saudi Arabia. Using a self-administered electronic questionnaire, a cross-sectional survey was conducted among domestic residents, who visited the mosque between 31 July and 3 August 2020. Participants’ demographic data, hand hygiene knowledge, perception, and practices were collected. Four hundred participants aged 18–65 (median 36) years completed the survey, of which 215 (53.8%) were female. The visitors’ mean knowledge score about hand hygiene was 6.4 (± standard deviation (SD) 1.35) of total 12. Most participants (392, 98%) were aware of the role of hand hygiene in preventing Coronavirus Disease 2019 (COVID-19); nevertheless, 384 (96%) said hand hygiene lowers body immunity and 316 (79%) thought <60% alcohol is sufficient for hand disinfection. Males had a higher knowledge score than females (6.46 (±1.41) vs. 6.14 (±1.27), p = 0.02) and, visitors who had no formal education scored higher than those with post-graduate education (6.88 (±1.45) vs 5.73 (±1.12), p = 0.01). Washing hands with soap and water was the predominant method practiced after a meal (365, 91.7%), after toilet visit (354, 88.5%), after touching a surface (262, 65.7%), after waste disposal (332, 83.2%), and when hands were visibly dirty (357, 89.5%). Al Madinah visitors had moderate knowledge about hand hygiene, but demonstrated some knowledge gaps and negligence in practice that are crucial to curb the spread of COVID-19.