Risk of neonatal mortality secondary to infections such as pneumonia and diarrhoeal diseases can be influenced by maternal hygiene, but little information is known about current maternal hygiene practices in low- and middle-income countries. This study aimed to assess the hygiene practices of mothers of neonatal intensive care unit (NICU) patients in Lusaka, Zambia and associated epidemiological factors. A cross-sectional survey of the mothers of hospitalized neonates at the University Teaching Hospital (UTH) in Lusaka was conducted. Study nurses orally administered questionnaires to mothers in their local languages about their hygiene practices related to newborn care. Of the 201 mothers surveyed, self-reported hygiene practice was associated with literacy (p = 0.013) and income (p < 0.0001). In contrast, adherence to recommended hygienic newborn care was less common, with only 36% of mothers practising recommended cord care practices. Forgetfulness (32%) and lack of hygiene resources (13%) were the major barriers to hygienic behaviours. Surveyed Zambian women who delivered babies requiring NICU care self-reported adequate hygienic practices and apparently faced difficulty practising them due to forgetfulness and a lack of hygiene resources. Future efforts should focus on improving cord care education, reinforcement of the importance of hygienic behaviours, and the supply of affordable antiseptic tools.