Background: This study has assessed health-related quality of life (HRQoL) and psychological distress between public sector professional groups. Methods: Short Form-36 Health Survey (SF-36) and Hospital Anxiety and Depression Scale (HADS) were administered to healthcare personnel, schoolteachers, and municipality and regional personnel in the region of Western Greece. Mean scores on all SF-36 dimensions and HADS were compared among these professional groups as well as with the Greek national norms to assess if there were significant differences between our study sample and the general population. Results: Healthcare personnel reported significantly lower SF-36 scores than teachers and municipality employees. Women reported poorer HRQoL than men on all SF-36 dimensions. This overall low score for health care workers masks the fact that male health care workers, primarily medical doctors, actually scored better than women, primarily nurses and auxiliary personnel. Average mean scores on all SF-36 dimensions reported by nurses and auxiliary personnel in healthcare were considerably lower than the ones from employees in all other occupational types. The impact on HRQoL observed mainly in vitality, social functioning and mental health was important. Many participants have shown psychological burden and depression. Conclusions: The health inequalities among healthcare employees are significant. This study is important to suggest taking measures for improving the HRQoL of health workers.