This dataset includes faecal lipid biomarker data from a lake sediment core from central Alaska and 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. The lake sediment core was recovered from Chisholm Lake in the Tanana Valley of interior Alaska (64.302267 N, 146.687331 W) in September 2019 using a Bolivia corer. Lead-210 (210Pb) measurements were completed by Flett Research and radiocarbon (14C) measurements were completed by the Center for Applied Isotope Studies at the University of Georgia. We extracted lipid biomarkers from 15 core subsamples and 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.