Introduction. Healthcare workers are more vulnerable to negative influences of the Covid-19 pandemic than cross-border travelers as they experience greater occupational stress, exacerbated by the possibility of self-contamination or contamination of their relatives through direct contact with patients, reduced social communication, deterioration in self-care and quality of life due to lack of energy and time. The aim of the study was to assess the level of stress and quality of life in healthcare workers of non-COVID-19-designated hospitals in Ukraine during the COVID-19 pandemic. Materials and Methods. There was carried out a case-control study that included 150 respondents divided into three groups: experimental groups (EG) 1 and 2 comprised 50 physicians and 50 nurses of non-COVID-19-designated hospitals, respectively; the control group (CG) included 50 healthy individuals not working in the healthcare field. The data were collected using paper questionnaires involving sociodemographic questions, the Perceived Stress Scale, and the 36-Item Short Form Health Survey. Results. EG 1 and EG 2 had significantly increased stress levels as compared to the CG. Physical functioning criterion in EG 2 was significantly lower than those in EG 1 and the CG. Vitality and social functioning indicators were significantly lower in EG 1 and EG 2 as compared to the CG. Quality of mental health was worse in EG 1 than in the CG. Conclusions. During the COVID-19 pandemic, healthcare workers of non-COVID-19-designated hospitals experienced a significant reorganization of their work environment, which required them to adjust rapidly to new conditions and psychological difficulties. The medical staff was found to have an increased level of perceived stress and decreased quality of life, which could affect the quality of medical care. Our findings suggest that the COVID-19 pandemic necessitates the development and implementation of stress coping measures to improve mental and physical health of healthcare workers, which can be used to achieve proper work and rest routines, create a microclimate within the team, and restore the work-life balance.