Background. Healthcare-associated infections (HAIs) cause substantial morbidity, mortality and healthcare costs. The prevalence of neonatal/paediatric HAI at South African (SA) district and regional hospitals is unknown.Objectives. To document HAI rates, antimicrobial use for HAI, infection prevention staffing, hand hygiene (HH) provisions and HH compliance rates in neonatal and paediatric wards in two district and two regional hospitals in the Western Cape Province, SA.Methods. An HAI point prevalence survey (PPS) was conducted in neonatal and paediatric wards at two district and two regional hospitals in the Western Cape during December 2016, applying National Healthcare Safety Network HAI definitions. HAI events and antimicrobial therapy active at 08h00 on the PPS day and during the preceding 7 days (period prevalence) were documented. Provisions for HH and HH compliance rates were observed on each ward using the World Health Organization’s HH surveillance tool.Results. Pooled point and period HAI prevalence were 9.9% (15/151; 95% confidence interval (CI) 6 - 15.8) and 12.6% (19/151; 95% CI 8 - 18.9), respectively. Hospital-acquired pneumonia (5/15, 33.3%), bloodstream infection (3/15, 20.0%) and urinary tract infection (3/15, 20.0%) were predominant HAI types. Risk factors for HAI were a history of recent hospitalisation (8/19, 42.1% v. 17/132, 12.9%; p<0.001) and underlying comorbidity (17/19, 89.5% v. 72/132, 54.5%; p<0.004). HH provisions (handwash basins/alcohol hand rub) were available and functional. HH compliance was higher in neonatal than in paediatric wards (125/243, 51.4% v. 25/250, 10.0%; p<0.001). Overall HH compliance rates were higher among mothers (46/107, 43.0%) than nurses (73/265, 27.8%) and doctors (29/106, 27.4%).Conclusions. Neonatal and paediatric HAIs are common adverse events at district and regional hospitals. This at-risk population should be prioritised for HAI surveillance and prevention through improved infection prevention practices and HH compliance.