This dataset includes modern faecal biomarker reference data from Alaskan herbivores and omnivores/carnivores. The purpose of this study was to assess the sensitivity and validity of lacustrine faecal steroid reconstructions of human and animal presence within the landscape by combining analyses of modern faecal samples and a sediment archive from Chisholm Lake, Alaska, where historical and archaeological population changes are well documented. To achieve this, we analysed faecal steroids (stanols and bile acids), which are biomolecules that are deposited in the landscape from faeces of higher vertebrates. We collected fresh modern-faecal samples from the Interior and Northern Alaska in spring-summer between 2017 and 2024 from moose (Alces alces), elk (wapiti; Cervus canadensis), plains bison (Bison bison bison), caribou (Rangifer tarandus), wood bison (Bison bison athabascae), sandhill crane (Antigone canadensis), mallard (Anas platyrhynchos), American black bear (Ursus americanus) and wolf (Canis lupus). We extracted lipid biomarkers from 50 modern faecal reference samples and, following saponification and column chromatography, faecal biomarkers were identified and quantified by GC-MS at the University of Bristol. Stanol and bile acid data are reported as and dry matter concentrations.