Cortisol is known to reflect the level of the stress response. However, measuring the cortisol concentration only once fails to provide sufficient information about the duration of the stress exposure. Moreover, handling is an acute stressor and increases cortisol secretion especially in wildlife species. Yet, in phocids reliable indicators are missing that reflect potential chronic effects of stress. The adrenal-derived steroid hormone dehydroepiandrosterone (DHEA) has been described as biomarker for the assessment of the stress status but has not been applied to marine mammal stress research. Therefore, DHEA, the sulfated precursor DHEAS and the cortisol/DHEA ratio were determined in serum of different seals. One group consisted of harbor (Phoca vitulina) and gray seals (Halichoerus grypus) that were habituated to human handling. The other two groups included healthy and free-ranging seals suffering from a disease. Blood samples were taken from 11 habituated (six males, five females), 17 wild_healthy (13 males, four females), and nine wild_diseased seals (four males, five females). No differences in serum cortisol levels could be detected between wild_healthy and wild_diseased seals. On the contrary, wild_diseased seals showed the lowest DHEA concentration compared to the other two groups. Wild_diseased seals also revealed the highest cortisol/DHEA ratio compared to the habituated_zoo (2,074.7 ± 351.4 vs. 121.5 ± 26.8, P < 0.001) and wild_healthy seals (827.9 ± 214.3, P < 0.01). DHEA and the cortisol/DHEA ratio may reflect differences in the functionality of the hypothalamic–pituitary–adrenal axis and therefore represent valuable tools for the assessment of stress-related effects in seals.