Measurements of cortisol levels in hair are a non-invasive method to study potential chronic stress that may affect carnivores' welfare. Using hair from 15 frozen and 18 taxidermied road-kill individuals, we aimed to provide information on the long-term physiological response of European wildcats (Felis silvestris silvestris) to habitat fragmentation and potential interspecific competition with golden jackals and red foxes. Our findings revealed that wildcats seemed to be unaffected by habitat fragmentation, suggesting that the facultative specialist behaviour of the species may lead to better toleration of human-altered environments. Red fox presence did not affect cortisol levels. However, significantly higher cortisol levels were measured in hairs of wildcats exposed to golden jackals, suggesting that the potential competition between the two species may lead to an increase in allostatic load in wildcats.