Healthcare-associated infections (HAIs) remain a serious threat to patient safety worldwide, particularly in low- and middle-income countries. Reducing the burden of HAIs through the observation and enforcement of infection prevention and control (IPC) practices remains a priority. Despite growing emphasis on HAI prevention in low- and middle-income countries, limited evidence is available to improve IPC practices to reduce HAIs. This study examined the perspectives of healthcare providers (HPs) and mothers in the neonatal intensive care unit on HAIs and determined the major barriers and facilitators to promoting standard IPC practices. This study draws on data from an ethnographic study using 38 in-depth interviews, four focus group discussions and participant observation conducted among HPs and mothers in neonatal intensive care units of a secondary- and tertiary-level hospital in Ghana. The qualitative data were analysed using a grounded theory approach, and NVivo 12 to facilitate coding. HPs and mothers demonstrated a modest level of understanding about HAIs. Personal, interpersonal, community, organizational and policy-level factors interacted in complex ways to influence IPC practices. HPs sometimes considered HAI concerns to be secondary in the face of a heavy clinical workload, a lack of structured systems and the quest to protect professional authority. The positive attitudes of some HPs, and peer interactions promoted standard IPC practices. Mothers expressed interest in participation in IPC activities. It however requires systematic efforts by HPs to partner with mothers in IPC. Training and capacity building of HPs, provision of adequate resources and improving communication between HPs and mothers were recommended to improve standard IPC practices. We conclude that there is a need for institutionalizing IPC policies and strengthening strategies that acknowledge and value mothers' roles as caregivers and partners in IPC. To ensure this, HPs should be better equipped to prioritize communication and collaboration with mothers to reduce the burden of HAIs.