In conservation strategies, getting precise and repeatable information on the species’ diet and health without relying on invasive or laborious methods is challenging. Here, we developed an efficient and non-invasive workflow for the sequencing and analysis of four taxonomic markers from fecal DNA to characterize the gut microbiota, parasites, and plants and lichens composing the winter diet of caribou (Rangifer tarandus), Canada's most iconic endangered species. Sequencing of the 18S rRNA gene of eukaryotes from seven locations in Manitoba and Saskatchewan, Canada, allowed for the detection of several parasites in caribou feces but provided limited information about plants and lichens - only algal symbionts were detected. An improved sensitivity and higher taxonomic resolution for plants and lichens was achieved through the sequencing of the ITS2 region, which revealed a rich winter diet in caribou as well as differences among sampling locations. Sequencing of the 16S rRNA gene of prokaryotes highlighted the relationships between the gut microbiota and both the diet and parasites. Overall, our results showed the potential of this multi-marker DNA metabarcoding workflow as an efficient tool to generate relevant information on the diet and health of caribou populations and provide insights into the species biology and ecology.