In this longitudinal study the effectiveness and outcomes of the Australian National Hand Hygiene Initiative (NHHI) were assessed. From 2009 to 2017 about 2 mio NHHI online learning programs were completed. The average hand hygiene compliance in Australian hospitals increased from 63.6% to 84.3% while the incidence of healthcare-associated Staphylococcus aureus bacteraemia (HA-SAB) decreased. For every 10% of increase in hand hygiene compliance, the incidence of HA-SAB decreased by 15%.