Environmental DNA-based diversity studies have increased in popularity with the development of high throughput sequencing technologies. This permits the potential simultaneous retrieval of vast amounts of molecular data from many different organisms and species, thus contributing to a wide range of biological disciplines. Most often, eDNA protocols designed for protists are focused on a small subunit of the ribosome gene, a highly conserved gene that does not permit species-level assignments. Consequently, the biological information associated species is lost, and the classification of the molecular diversity into Operational Taxonomic Units cannot be compared directly between studies. To solve this issue, specific eDNA protocols based on fast evolving genes applicable to the species level have been proposed. However this approach is not yet applied to protist metabarcoding, except in marine and shallow lotic freshwater ecosystems. Therefore in this study, we present a species-level eDNA protocol, designed to explore diversity of Arcellinida (Amoebozoa: Tubulinea) testate amoebae taxa, that is based on mitochondrial cytochrome oxidase subunit I (COI). These organisms are widespread in lentic water bodies and soil ecosystems. We applied this protocol to 42 samples from peatlands, estuaries and soil environments, recovering all the infraorders in Glutinoconcha (with COI data) except for Hyalospheniformes. With this protocol we expect to revolutionize the design of modern distributional Arcellinida surveys. Through an approach that involves rapid analysis, is cost effective and permits reliable handling of vast sampling designs, Arcellinida have to potential to be established as a model group for an array of theoretical and applied studies.