Background: Human Nipah virus (NiV) infection is an emerging zoonotic disease caused by the NiV resulting in severe illness in humans. The physicians can represent a lead role in disease prevention if they have the right knowledge regarding disease. This study aimed to state the assessment of knowledge regarding NiV infection among physicians in a selected tertiary hospital, Rangpur, Bangladesh.Methods: A cross-sectional study was conducted among 211 physicians in Rangpur Medical College and Hospital by pretested structured questionnaire, from January 2020 to December 2020, using a convenient sampling method. Data were collected through face-to-face interviews.Results: The majority of the respondents (69%) were within the 21 to 25 years of age group, mean age was 25±2.9 years where 54% of respondents were female. A questionnaire was comprised of 87 questions regarding knowledge on NiV infection. The findings revealed that 19% had good knowledge, 50% had fair and about 31% had poor level of knowledge regarding NiV infection. Among the respondents, 83% mentioned lack of awareness as a barrier regarding the prevention of NiV infection. Inferential statistics were done at a 95% confidence interval and 5% level of significance. Those who were aged between 21 to 25 years had significantly good knowledge than those who were more than 26 years of age (p=0.002).Conclusions: This study concludes that knowledge of the physicians on NiV infection was at a fair or average level. There is a dire need for the routine integration of the awareness and safety precaution practice among the physicians.