Animals face several challenges in their natural environment, and to cope with such conditions, they may exhibit contrasting physiological responses that directly affect their overall well-being and survival. In this study, we assessed physiological responses via faecal glucocorticoid metabolite (fGCM) measurements in free-ranging mugger crocodiles inhabiting diverse habitats in Gujarat, India. We sampled muggers within Charotar, a rural area (Zone A) with local people having high tolerance towards the presence of muggers, and Vadodara, a region having both urban (Zone B) and rural (Zone C) areas with high levels of human–mugger conflict (HMC). Further, muggers in Vadodara live in water bodies that are mostly polluted due to sewage disposal from adjoining chemical industries. To measure fGCM (mean ± SEM, ng/g dry faeces) levels in muggers, scats were collected during both breeding (N = 107 scats) and non-breeding (N = 22 scats) seasons from all three zones. We used captive muggers (a focal enclosure) to biologically validate (via capture and restraint) the selected fGCM assay (11-oxoetiocholanolone assay). We showed a significant (P < 0.05) 11-fold increase in fGCM levels between pre-capture (540.9 ± 149.2, N = 11) and post-capture (6259.7 ± 1150.5, N = 11) samples. The validated assay was applied to free-ranging muggers during the breeding season, and Zone A showed significantly (P < 0.05) lower fGCM levels (542.03 ± 71.3) compared to muggers of Zone B (1699.9 ± 180.8) and Zone C (1806.4 ± 243.2), both zones having high levels of HMC with polluted water bodies. A similar contrast in fGCM levels was also observed during the non-breeding season. Overall, the study demonstrated that fGCM levels in muggers varied across habitats, and such variation could be due to a multitude of ecological factors that the species experience in their immediate local environment. Moreover, high fGCM levels in muggers of Vadodara during both breeding and non-breeding seasons may indicate a condition of chronic stress, which could be maladaptive for the species.