Purpose: To evaluate the healthcare workers’ (HCWs) knowledge towards Nipah virus and identify the associated variables with good knowledge. Methods: A cross-sectional design was conducted in Najran, Kingdom of Saudi Arabia (KSA) to evaluate the knowledge of HCWs towards Nipah virus. A validated questionnaire was employed to collect the data of HCWs. It consisted of two parts: Socio-demographic characteristics of the participants and questions related to the knowledge towards Nipah virus. Percentages and median (Q1, Q3) were used to present the data and were compared by Mann–Whitney and Kruskal Wallis. The associated variables with good knowledge were identified by logistic regression. Results: The study included 247 participants. The median (Q1, Q3) knowledge score was 45% (10%, 60%), reflecting poor knowledge. About 62% of the HCWs stated that the symptoms of Nipah virus infection could be acute respiratory distress, convulsions, and coma. Only 36.8% reported no available vaccine to prevent Nipah virus infection, and more than 50% identified the virus’s main reservoir (fruit bat) as the possibility of transmission from animal to human. Roughly 57% of them reported that the virus can be transmitted among people through droplets. Less than 40% stated the Nipah virus can cause AIDS. The significantly associated variable with good knowledge was only nationality. Conclusion: The present study showed a poor knowledge of HCWs. Good knowledge was associated with nationality. Therefore, the implementation of education and training programmes are highly recommended throughout conducting prospective and interventional studies.