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Purpose - Neonatal mortality rate is 24 deaths per 1,000 live births in Bangladesh and most of them die during early neonatal period. The purpose of this paper is to assess the knowledge and attitude of neonatal care practices among postnatal mothers in a tertiary care hospital in order to provide a basis for the development of strategies to improve further. Design/methodology/approach - A cross-sectional study was carried out in the Dhaka Medical College Hospital, Bangladesh using a convenient sampling technique. In total 211 postnatal mothers were interviewed using a structured questionnaire and χ2 test was used to analyze the data. Findings - Among mothers, 37.9 percent were aged between 16–20 years; 16.1 percent had no institutional education; 55 percent were primipara and only 26.5 percent had attended antenatal visit for more than four times. Mothers had apparently good knowledge and attitude about thermoregulation, early initiation of breast-feeding, importance of providing colostrum to the newborn, exclusive breast-feeding (EBF) up to six months of age and immunization at birth. However, this study identified knowledge gap about cord care, eye care, first bathing and hygiene practices. Overall, only a small proportion of respondents had good level of knowledge (37 percent) and attitude (47.4 percent) on newborn care. Highly significant statistical association was found between the knowledge, attitude level and socio-demographic characteristics of respondents. Knowledge and attitude were strongly associated as well. Research limitations/implications - There is scope for improvement by providing health education to antenatal and postnatal mothers. Therefore, implementation of the guidelines outlined in the Maternal and Child Handbook (MCH) is highly recommended. Originality/value - There is scope for improvement by providing health education to antenatal and postnatal mothers. Therefore, implementation of the guidelines outlined in the MCH is highly recommended to enhance the knowledge and attitude on newborn care among postnatal mothers.