The measurement of hair cortisol is increasingly used to understand the effect of natural and anthropogenic stressors on wild animals, but it is potentially confounded by individual, seasonal and sex-dependant variations in baseline cortisol secretion. This study validated an enzyme-linked immunoassay for hair cortisol measurement and characterized its baseline variation in a wild population of Egyptian mongoose. The analysis encompassed individuals of both sexes and all ages, across a range of geographic, environmental and seasonal conditions that the species experiences in Portugal allowing us to account for spatial, temporal and biological factors that contribute to hair cortisol variation. Our results showed that age, sex and storage time had an effect on hair cortisol, but season did not. Hair cortisol was higher in early stage juveniles compared to other age cohorts, in males when compared to females, and decreased with longer storage time. By identifying the factors that influence baseline hair cortisol in this wild population, we establish the basis for its application as an indicator of the effect of natural and anthropogenic stressors.