AIM The aim of this cross-sectional, descriptive study was to compare assessments and relationships of stress of conscience, perceptions of conscience, burnout and social support between healthcare personnel (HCP) working in two different organisations for care of older people. METHODS This cross-sectional, descriptive comparative study was performed among Registered Nurses and nurse assistants working in two different organisations (n(1) = 98, n(2) = 488) for residential care of older people. The organisations were chosen to be as different as possible, and data were collected using four different questionnaires. Hierarchical cluster analysis with multiscale bootstrap resampling was used to compare the associations between all items in the questionnaires. Descriptive statistics, 95% confidence intervals, chi-squared tests, Cohen's d, Cramer's V and the φ coefficient were all used to judge differences between the organisations. RESULTS The associations between stress of conscience, perceiving one's conscience as a burden, and burnout were similar in both organisations. Perceiving one's conscience as far too strict and having a troubled conscience from being unable to live up to one's standards were associated with stress of conscience and burnout in one organisation. Women had higher levels of stress of conscience and reported lower social support from co-workers compared with men. CONCLUSIONS This study shows that associations between perceptions of conscience, stress of conscience and burnout are common experiences that are similar among HCP despite great differences in the characteristics of organisations. It can be burdensome for HCP to be unable to realise their ambitions to provide good care, and sex/gender can be an important factor to consider in the development of measures against the negative effects of stress of conscience. More studies are needed about how HCP's ambition to provide good care and sex/gender are related to perceptions of conscience, stress of conscience and burnout.