Background: The National Hand Hygiene Initiative (NHHI) is a standardised culture-change programme based on the WHO My 5 Moments for Hand Hygiene approach to improve hand hygiene compliance among Australian health-care workers and reduce the risk of health-care-associated infections. We analysed its effectiveness. Methods: In this longitudinal study, we assessed outcomes of the NHHI for the 8 years after implementation (between Jan 1, 2009, and June 30, 2017), including hospital participation, hand hygiene compliance (measured as the proportion of observed Moments) three times per year, educational engagement, cost, and association with the incidence of health-care-associated Staphylococcus aureus bacteraemia (HA-SAB). Findings: Between 2009 and 2017, increases were observed in national health-care facility participation (105 hospitals [103 public and two private] in 2009 vs 937 hospitals [598 public and 339 private] in 2017) and overall hand hygiene compliance (36 213 [63·6%] of 56 978 Moments [95% CI 63·2–63·9] in 2009 vs 494 673 [84·3%] of 586 559 Moments [84·2–84·4] in 2017; p<0·0001). Compliance also increased for each Moment type and for each health-care worker occupational group, including for medical staff (4377 [50·5%] of 8669 Moments [95% CI 49·4–51·5] in 2009 vs 53 620 [71·7%] of 74 788 Moments [71·4–72·0]; p<0·0001). 1 989 713 NHHI online learning credential programmes were completed. The 2016 NHHI budget was equivalent to AUD$0·06 per inpatient admission nationally. Among Australia's major public hospitals (n=132), improved hand hygiene compliance was associated with declines in the incidence of HA-SAB (incidence rate ratio 0·85; 95% CI 0·79–0·93; p≤0·0001): for every 10% increase in hand hygiene compliance, the incidence of HA-SAB decreased by 15%. Interpretation: The NHHI has been associated with significant sustained improvement in hand hygiene compliance and a decline in the incidence of HA-SAB. Key features include sustained central coordination of a standardised approach and incorporation into hospital accreditation standards. The NHHI could be emulated in other national culture-change programmes. Funding: Australian Commission on Safety and Quality in Health Care.