This observational study measured healthcare workers’ (HCWs’), patients’ and visitors’ hand hygiene compliance over a 24h period in two hospital wards using the ‘five moments of hand hygiene’ observation tool. Hand hygiene is considered to be the most effective measure in reducing healthcare-associated infections but studies have reported suboptimal levels of compliance. Most studies have used random observational time-periods for data collection and this has been criticised. We monitored a total of 823 hand hygiene opportunities (HCWs, N=659; patients and visitors, N=164). Among HCWs, compliance was 47% for doctors, 75% for nurses, 78% for allied health professionals, and 59% for ancillary and other staff (P<0.001). There was no difference in compliance between patients and visitors (56% vs 57%, P=0.87). Hand hygiene compliance varied depending on which of the five moments of hygiene HCWs had undertaken (P<0.001), with compliance before an aseptic task being 100% (3/3); after body fluid exposure 93% (86/93); after patient contact 80% (114/142); before patient contact 68% (196/290); and after contact with surroundings 50% (65/129). Lower levels of compliance were found for HCWs working during the early shift (P<0.001). For patients and visitors there was no evidence of an association between moments of hygiene and compliance. Levels of compliance were higher compared with previous reported estimates. Medical staff had the lowest level of compliance and this continues to be a concern which warrants specific future interventions.