The impact of the COVID-19 pandemic on the mental health of healthcare professionals is currently under research and prevalence of mental health symptoms across the world vary a lot. Moreover, knowledge and perceptions of healthcare professionals towards the new coronavirus is yet to be explored since very few data have been published to date. Thus, we decided to conduct a cross-sectional, web-based survey to measure the levels of depressive, anxiety and stress symptoms using the Depression, Anxiety and Stress Scale-21. The knowledge and perceptions of healthcare professionals towards the new coronavirus were also examined with a self-constructed questionnaire. Data were collected between April 19th and May 31st 2020. In total, 1484 professionals participated in the survey and 1064 completed it in full; 60.8% were females, 66.5% were physicians and 24.3% were first-line healthcare workers. The prevalence of at least moderate symptoms was 13% for depression, 11.9% for anxiety, and 11.3% for stress. Women, younger participants, residents in urban areas, having lower income and worse self-reported health status had higher scores in all outcomes. First-line healthcare workers also indicated higher anxiety scores compared to those who were not first responders. Regarding knowledge and perceptions, most participants agreed with the asymptomatic nature of the virus and its heightened danger for older individuals and those with underlying health conditions. Different views were expressed regarding the possibility of airborne transmission, its similarity to common flu, and the statements that the new coronavirus is manufactured and serves a specific purpose and that it is out of control. In conclusion, the results of our study suggest that the prevalence of depressive, anxiety and stress symptoms in Greek healthcare professionals is placed in the lower end of the range reported from various recent studies across the world. Nevertheless, professionals at risk should be monitored closely and supported when needed.