Measurement of faecal glucocorticoid metabolites is increasingly used as a non-invasive tool to examine disturbances in various domestic and wild animals. Because measurements of faecal glucocorticoid metabolites has previously never been reported in fallow deer, we determined 11,17-dioxoandrostanes (11,17-DOA), a group of cortisol metabolites, in the faeces of four fallow deer yearlings after an adrenocorticotropic hormone (ACTH) challenge or control saline injection by an 11-oxoaetiocholanolone enzyme immunoassay (EIA), to validate a method. A 2.9- to 4.3-fold increase in measured cortisol metabolites in challenged animals after approximately 22 h demonstrated the suitability of this group-specific EIA to monitor adrenocortical activity in respective deer species. To determine faecal cortisol metabolites in fallow deer from a Mediterranean habitat, we collected samples during a 1-year study at Veliki Brijuni Island. The study confirmed seasonal pattern of cortisol release in fallow deer. Higher 11,17-DOA concentrations (median; min-max) were determined for November (99; 50-2,035), March (112; 25-315) and May (92; 40-196 ng/g faeces). Significantly lower concentrations were measured during July (30; 10-195 ng/g faeces). This study indicates that the analysis of faecal glucocorticoid metabolites is a valuable non-invasive technique for monitoring adrenocortical activity in fallow deer. This, together with information about the seasonal pattern of glucocorticoid excretion, could help to improve fallow deer management and welfare, especially in the case of farmed and park animals.