The COVID-19 pandemic has significantly affected the mental health of healthcare workers, who have taken on the major problems triggered by the emergency. The mental consequences concern high levels of insomnia, anxiety, depression and burnout, which inevitably affect their professional quality of life too. The aim of this study was to analyze the relationship between psychopathological symptoms (tested with the Depression Anxiety Stress Scale, DASS-21) and professional quality of life (measured with the Professional Quality of Life Scale, ProQol) in a hospital of southern Italy. 204 healthcare workers were recruited by non-probabilistic sampling and divided by age, gender, work roles (physicians, nurses and intermediate care technicians) and clinical departments (Cardio-medicine, Infectious Diseases, Emergency Medicine, First Aid, Obstetrics and Pneumology). The results showed higher levels of Secondary Traumatic Stress, Depression, Anxiety and Stress in women than in men. Physicians and nurses experienced lower levels of Compassion Satisfaction but higher Burnout than intermediate care technicians; likewise, nurses were more anxious than physicians. The Emergency Medicine had higher scores in Compassion Satisfaction than Infectious Disease, Pneumology, Obstetrics and Cardio-Medicine. In light of what has been said so far, it appears essential to intervene on the first mild signs of Burnout and Secondary Traumatic Stress, because they precede the onset of Depression, Stress and Anxiety in healthcare workers.