Ecosystems with high natural value (HNV) have generally been maintained by agricultural practices and are increasingly important for the ecosystem services that they provide and for their socio-economic impact in the ever-changing context. Biodiversity conservation is one of the main objectives of the European Green Deal, which aims to address biodiversity loss, including the potential extinction of one million species. The aim of this research was to trace the effects of organic and mineral fertilizers on the floristic composition, but also on the number of species, of the grasslands with high biodiversity (HNV) from the Transylvanian Plain, Romania. The experiments were established in 2018 on the nemoral area and analyzed the effect of a gradient of five organic and mineral treatments. Fertilization with 10 t ha−1 manure or N50 P25K25 ensures an increase in yield and has a small influence on diversity, and it could be a potential strategy for the maintenance and sustainable use of HNV grasslands. Each fertilization treatment determined species with indicator value that are very useful in the identification and management of HNV grasslands. The dry matter biomass increases proportionally as the amounts of fertilizer applied increase and the number of species decreases.