AIM: The outbreak of COVID-19 has laid unprecedented psychological stress on healthcare workers (HCWs). We aimed to assess the immediate psychological impact of COVID-19 epidemic on the HCWs at Baqiyatallah Hospital in Tehran, Iran. MATERIAL AND METHODS: We conducted a cross-sectional survey of HCWs using questionnaires in February and March 2020 in Baqiyatallah Hospital, Tehran. We evaluated depression, stress, and anxiety levels using the DASS-21 questionnaire. Participants were selected by using census sampling. All statistical analyses were performed using R version 3.5.1. RESULTS: The study population included 217 HCWs (111 male, 116 female) and the mean age of the study group was 39.6 years old. Approximately two-thirds of the HCWs stayed in the hospital for 2-3 weeks. The mean scores of depression and stress were at a "severe" level, while anxiety scores were at an "extremely severe" level. The prevalence of severe scores was 38.71%, 2.30%, and 48.97% for depression, anxiety, and stress, and the prevalence of extremely severe scores was 46.54%, 97.24%, and 4.98% depression, anxiety, and stress, respectively. In stress subscale, moderate stress was 47.46%. Female HCWs reported higher levels of depression compared with males. CONCLUSION: In this study, HCWs reported experiencing severe and extremely severe psychological burdens. Timely interventions to promote mental health in HCWs exposed to patients with COVID-19 need to be immediately implemented, with female nurses requiring particular attention. This process could be facilitated via tests for molecular biomarkers in accessible body fluids, such as saliva, plasma, and serum.