Habitat disturbance due to anthropogenic activities is a source of acute and chronic energetic stress in wild animals, including primates. Physiological responses to stress can compromise growth and reproduction, increase susceptibility to infection and lead to deleterious effects on health and conservation efforts. However, physiological measures of energetic stress in association with habitat disturbance are uncommon, especially for wild primate species. Here, we report differences in the stress hormone cortisol in two subpopulations of wild gray-cheeked mangabeys (Lophocebus albigena) inhabiting disturbed and undisturbed forest areas of Kibale National Park, Uganda. Cortisol levels were assessed via opportunistically and noninvasively collected urine samples using previously validated methods. We hypothesized that mangabeys in disturbed forest (DF) areas would experience greater stress and therefore exhibit higher average cortisol levels than conspecifics in nearby relatively undisturbed forest areas (UF). As predicted, mangabeys in the disturbed area had significantly higher cortisol levels (unpaired t-test of log transformed data, t = 4.88, d.f. = 108, P < 0.0001). Mangabeys in undisturbed forest exhibited expected diurnal patterns of cortisol excretion while those in disturbed areas did not, suggesting alteration of the circadian pattern of hypothalamic-pituitary-adrenal function (DF, r = 0.12, P = 0.43; UF, r = 0.35, P = 0.005). Reasons for differences are unclear, but could include altered food availability and distribution, human contact or other anthropogenic effects. Noninvasive measurements of urinary hormones are useful for quantifying animal energetic stress in the wild and assessing the effects of conservation efforts to attenuate anthropogenic stress in wild populations.