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 five genera of parasites in caribou feces (including Nematodirella and Parelaphostrongylus) with variable frequency of occurrence depending on sampling location and sex. Our workflow also revealed a rich winter plant and lichen diet in caribou, with respectively 29 and 18 genera identified across all samples through plant and fungal ITS2 sequencing. Relationships between the gut microbiota and both the diet and parasite richness were also identified. Of note, the Central Saskatchewan sampling location was characterized by a clearly distinct gut microbiota which could be linked to an epiphytic lichen-rich diet. Overall, our results showed the potential of this multi-marker DNA metabarcoding workflow as an efficient tool to provide insights into the species biology and ecology.