This study examined Hajj pilgrims’ knowledge and reported practice of hand hygiene. In Hajj 2019, a cross-sectional survey was undertaken in Mina, Makkah, Saudi Arabia, of domestic Saudi pilgrims aged ≥18 years by using a self-administered Arabic questionnaire that captured data on pilgrims’ socio-demographics, hand hygiene knowledge, and reported practices of hand cleaning following certain actions. A total of 348 respondents aged 18 to 63 (median 32) years completed the survey, of whom 200 (57.5%) were female. The mean (±standard deviation (SD)) hand hygiene knowledge score was 6.7 (±SD 1.9). Two hundred and seventy one (77.9%) and 286 (82.2%) of respondents correctly identified that hand hygiene can prevent respiratory and gastrointestinal infections respectively, but 146 (42%) were not aware that it prevents hand-foot-mouth disease. Eighty-eight (25.3%) respondents erroneously reported that hand hygiene prevents HIV. Washing hands with water and soap was the most preferred method practiced before a meal (67.5% (235/348)), after a meal (80.2% (279/348)), after toilet action (81.6% (284/348)), when hands were visibly soiled (86.2% (300/348)), and after waste disposal (61.5% (214/348)). Hajj pilgrims demonstrated a good knowledge and practice of hand hygiene, but there are gaps that are vital to control outbreaks such as COVID-19.