Introduction: Ebola is a viral infection with a high mortality risk and health workers are considered as an at-risk group. The main aim of this study was to determine the knowledge and attitude of health workers about Ebola virus disease (EVD) in Yazd city, Iran. Methods: This cross-sectional study was carried out on 110 health workers in health centres of Yazd city, Iran in 2015, who were selected by cluster random sampling method. The tool for data collection was a researcher-made questionnaire including demographic information as well as knowledge and attitude questions about EVD with confirmed validity and reliability. Data were analysed by statistical tests of Student’s T-test, Chi-square, ANOVA, and Pearson correlation coefficient. Results: The mean score of the participants' knowledge and attitude were 25.16± 3.58 (range 0-46) and 46.59±4.05 (range 13-65), respectively. More than half of the health workers did not know transmission routes of EVD. However, 59.6% of them were aware that the one way to prevent the disease was to avoid contact with an infected person. A significant correlation was also found between knowledge and attitude scores (P< 0.001, r= 0.384). Conclusion: Since knowledge of the health workers was at the moderate level, training courses and interventional programs are recommended for health care workers especially about transmission routes and signs of EVD to increase the Knowledge and attitude of the participants