Abstract Physiological biomarkers are commonly used to assess the health of taxa exposed to natural and anthropogenic stressors. Glucocorticoid (GC) hormones are often used as indicators of physiological stress in wildlife because they affect growth, reproduction and survival. Increased salinity from human activities negatively influences amphibians and their corticosterone (CORT; the main amphibian GC) physiology; therefore, CORT could be a useful biomarker. We evaluated whether waterborne CORT could serve as a biomarker of salt stress for three free-living amphibian species that vary in their sensitivity to salinity: boreal chorus frogs (Pseudacris maculata), northern leopard frogs (Rana pipiens) and barred tiger salamanders (Ambystoma mavortium). Across a gradient of contamination from energy-related saline wastewaters, we tested the effects of salinity on baseline and stress-induced waterborne CORT of larvae. Stress-induced, but not baseline, CORT of leopard frogs increased with increasing salinity. Salinity was not associated with baseline or stress-induced CORT of chorus frogs or tiger salamanders. Associations between CORT and salinity were also not related to species-specific sensitivities to salinity. However, we detected background environmental CORT (ambient CORT) in all wetlands and spatial variation was high within and among wetlands. Higher ambient CORT was associated with lower waterborne CORT of larvae in wetlands. Therefore, ambient CORT likely confounded associations between waterborne CORT and salinity in our analysis and possibly influenced physiology of larvae. We hypothesize that larvae may passively take up CORT from their environment and downregulate endogenous CORT. Although effects of some hormones (e.g. oestrogen) and endocrine disruptors on aquatic organisms are well described, studies investigating the occurrence and effects of ambient CORT are limited. We provide suggestions to improve collection methods, reduce variability and avoid confounding effects of ambient CORT. By making changes to methodology, waterborne CORT could still be a promising, non-invasive conservation tool to evaluate effects of salinity on amphibians.