Background: Globally, infections are the third leading cause of neonatal mortality. Predominant risk factors for facility-born newborns are poor hygiene practices that span both facilities and home environments. Current improvement interventions focus on only one environment and target limited caregivers, primarily birth attendants and mothers. To inform the design of a hand hygiene behavioural change intervention in rural Cambodia, a formative mixed-methods observational study was conducted to investigate the context-specific behaviours and determinants of handwashing among healthcare workers, and maternal and non-maternal caregivers along the early newborn care continuum. Methods: Direct observations of hygiene practices of all individuals providing care to 46 newborns across eight facilities and the associated communities were completed and hand hygiene compliance was assessed. Semi-structured interactive interviews were subsequently conducted with 35 midwives and household members to explore the corresponding cognitive, emotional and environmental factors influencing the observed key hand hygiene behaviours. Results: Hand hygiene opportunities during newborn care were frequent in both settings (n = 1319) and predominantly performed by mothers, fathers and non-parental caregivers. Compliance with hand hygiene protocol across all caregivers, including midwives, was inadequate (0%). Practices were influenced by the lack of accessible physical infrastructure, time, increased workload, low infection risk perception, nurture-related motives, norms and inadequate knowledge. Conclusions: Our findings indicate that an effective intervention in this context should be multi-modal to address the different key behaviour determinants and target a wide range of caregivers…more