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Introduction Healthcare professionals, due to the nature of their work, have always experienced occupational stress, depression and low quality of life, which have been aggravated during the COVID-19 pandemic. Aim A large-scale cross-sectional descriptive correlational study aimed to investigate the impact of the COVID-19 pandemic on Greek healthcare professionals’ psychological status and quality of life. Material and Methods The study was conducted at “Attikon” General University Hospital and the 2nd Health Region in Athens, Greece. An assessment of anxiety and depression was carried out using the Zung’s Self-Rating Anxiety and Depression Scale (SAS/SDS). To assess the participants’ Quality of Life (QoL) the Short Form Survey-36 (SF-36) was used. Results 147 healthcare professionals were enrolled in the study. 70.7% experienced normal stress levels, 23.8% mild, 4.8% moderate and 0.7% severe. Mild depression was experienced by 34.7%, moderate by 10.2% and severe by 1.4%, with a 53.7% showing no depressive symptoms. Women experienced higher levels of anxiety and depression (p=0.001 & 0.001 respectively), and were 5.4 times more at risk to develop anxiety [Odds Ratio (OR) 5.357, 95% Confidence Interval (CI), 1.95-14.72: p=0.001] and 3.4 depression (OR, 3.365, 95% CI, 1.59- 7.12: p=0.002). Nurses and other professionals experienced higher stress and depression levels (p=0.004 & 0.040 respectively) than doctors. Participants reporting more exhaustion exhibited higher anxiety and depression levels (p=0.001). Compared to the pre-COVID-19 era, women (p=0.001), other health professionals (p=0.001) and those experiencing more physical burnout during COVID-19 (p=0.005) reported worse physical health. Anxiety and depression were negatively correlated with most sub scales of SF-36 except social functioning and bodily pain (p=0.001). Conclusions Healthcare professionals’ QoL has been affected by the COVID-19 pandemic and they experience higher levels of anxiety and depression. There is a need to develop strategies to address the negative psychological impact of this pandemic on healthcare professionals.