Background: Worldwide mountain regions are recognized as hotspots of ethnopharmacologically relevant species diversity. The high plant diversity and isolated population of South Tyrol (Southern Alps, Italy) have prospered a unique traditional botanical knowledge of medicinal plants, which dates back to prehistoric times. However, changes in rural life and culture may threaten the unique biodiversity and cultural heritage. Our study aims to collect and analyze information on native plants used in traditional folk medicine, focusing on the preservation of botanical and cultural diversity.Methods: Data was collected by a review of published material documenting traditionally used medicinal plants of South Tyrol, to capture the total diversity of plants and their usage. We evaluated different parameters, comprising the Ethnobotanicity Index (EI), Ethnophytonomic Index (EPI), relative frequency of citation (RFC), Red List status, and regional legislation with regard to the plant species.Results: A total of 275 species, including 3 mushrooms and 3 lichens were identified, belonging to 72 families, most frequently to the Asteraceae, Rosaceae, and Lamiaceae. Most cited species were Hypericum perforatum L., Urtica dioica L., and Plantago lanceolata L. According to 12 ICPC-2 disease categories most frequently treated for human health were digestive and respiratory systems as well as the skin. A total of 27 species were listed as endangered, of which 16 are not protected and two are already extinct. Among the 59 predominantly Alpine species, 11 species are restricted to the high altitudes of the Alps and might be threatened by global warming.Conclusions: Our research revealed that ethnobotanical richness of South Tyrol is among the highest in Italy and throughout the Alps. Nevertheless, it is evident that biodiversity and traditional knowledge have been heavily eroded. Furthermore, we point out particularly sensitive species that should be reconsidered in legal regulations for stronger protection.