Background: Neonatal hospital infections, in addition to being the cause of a significant number of perinatal, neonatal, and postnatal deaths, are also associated with increased health care costs. This is because hospitalization of infected children is up to threefold longer than that of non-infected children. Objective: Our objective is to assess the nurse’s knowledge and observe their practice to prevent and control neonatal nosocomial infection in maternal and neonatal unit at Rajshahi Medical College Hospital. Results: Age distribution shows highest percentage 48 % in between 40-49 years. All of the respondents were female. Most of the nurses 90% were Muslim, 8% were Hindu. About 100% of the respondents have S. S. C., 30% have H. S. C. and 4% have B.A. / B.Sc. degree, Remaining 4% of the respondents having Masters or others degree. Regarding professional qualification 100% of respondent’s have diploma in nursing and midwifery, 20% of B.Sc. in public health, and remaining 14% have M.P.H. In service about 6% of respondent’s job experience was1-5years, 12% had 6-10 years and the 24% had 11-15 years, 20% had 16-20years, 10% had 21-25 years and remaining 28% had 26-30years experience. The grading of the knowledge was about 73.5% of nurses had complete knowledge, 15% had partial knowledge, 5.33% gave wrong answer and 6.16% said that they do not know the answer. About 50% of the neonatal age was 5- 10 days, about 20% of 11-16 days, nearly 18% was 17-22 days, and the remaining 12% was 23-28 days. Among them 56% was male and 44% was female. Among the responding parents, family type was mainly nuclear 62% and joint 38%. Educational qualification of the father’s was about 22% illiterate, 20% able to write their name, 18% was primary educated, 20% was secondary,14% higher secondary and 6% was graduate. On the other hand mother’s educational qualification was about 30% illiterate, 20% was able to write their name, primary education complete about 24% and secondary 18% and higher secondary 8%.According to the data collection the respondents residence in the Rajshahi division was 100%, residence in the rural 70% and urban about 30%. Conclusion: Current global evaluations confirm that commitment to improving newborn health makes meaningful socio-economic contributions. Various reasons can be attributed to why the health of the newborn has been neglected despite the huge mortality rates and why most neonatal deaths are unseen and undocumented.