Health care workers (HCWs) face a higher risk of infection, since they work at the front line of COVID-19 patients' management. Misinterpretations of current scientific evidence among HCWs may impact the delivery of appropriate care to COVID-19 patients and increase the risk of SARS-CoV-2 transmission in the hospital setting. Moreover, knowledge may affect HCWs perceptions depending on their broad beliefs and past experiences. The aim of this study was to explore the knowledge and perceptions of HCWs regarding COVID-19 issues during the second wave of the pandemic. A cross-sectional survey, involving a printed questionnaire, was conducted from 21 October 2020 to 31 January 2021 in four tertiary care hospitals located at four distant geographical regions in Greece. In total, 294 HCWs participated in this study. The majority of HCWs provided precise responses regarding general knowledge, perceptions, and practices concerning the COVID-19 pandemic. However, responses on hand hygiene and antimicrobial use in HCWs with COVID-19 were mistaken. This study reveals a certain degree of misconceptions and knowledge gaps in HCWs everyday practice, especially regarding hand hygiene and antimicrobial use in COVID-19 patients.