In the current Ebola virus disease (EVD) outbreak, health care workers (HCWs) are on the frontline of the response. The study aimed to report on the knowledge, attitudes and practices of Conakry HCWs. A cross-sectional survey was done among HCWs from the two national hospitals and at the communal hospitals in Conakry, using a standardized questionnaire. Demographic, knowledge, attitudes and practices data of the respondents were recorded and analyzed. Respondents (n = 525) were mainly physicians (43.0%) or nurses (36.8%). Over 90% of HCWs cited the three major clinical signs of EVD. Only 68% knew the correct concentration of Javel water that is advised for hand washing. This was known by 69.4% of nurses, 59.0% of residents, 69.5% of physicians, 100% of pharmacists and 60.9% of midwifes (P = 0.35). The EVD transmission risk in the ward was estimated as probable for 70.6% and very probable for 24.0% of HCWs. Eighty-four percent of all respondents estimated their knowledge on EVD insufficient and 97.1% reported that they either need specific training on EVD or enhance their knowledge. This study underlines the low level of knowledge, attitude and perceptions regarding EVD prevention despite the high incidence and mortality of this disease.