Background/Aims Neonatal infection is one of the most common health challenges for newborn care globally. It is also a common contributory reason for admission to neonatal intensive care units in low- and middle-income countries and one of the main causes of mortality and morbidity. This study aimed to assess mothers’ knowledge, practice and perception of factors affecting infection prevention. Methods A descriptive cross-sectional study was conducted among 193 neonatal mothers from August to September 2021. A structured questionnaire was used to collect data on knowledge and practice of infection prevention, as well as factors the participants felt impacted infection prevention. Data were analysed using descriptive statistics. Results The majority of mothers had good knowledge (93.3%) and practice (92.7%) of infection prevention. The participants reported multiple factors that they felt impacted neonatal infection prevention, including the mother's education (84.5%), home birth practices (89.1%), poor economic state (85.0%), poor antenatal attendance (93.3%), negative cultural and belief systems (94.8%) and a lack of knowledge of infection prevention (90.7%). Conclusions Overall, the majority of neonatal mothers had good knowledge and practice of infection prevention. The Ghana Health Service and Ministry of Health should encourage women to give birth at a health facility, to increase their knowledge of infection prevention, which will then be reflected in improved practice.