New applications of DNA and RNA sequencing are expanding the field of biodiversity discovery and ecological monitoring, yet questions remain regarding precision and efficiency. We used a set of documented mock communities comprising 13 species of freshwater macroinvertebrates of estimated individual biomass, to compare the detection efficiency of COI metabarcoding (3 different amplicons) and shotgun mito-metagenome sequencing. Additionally, we used individual COI barcoding and de novo mitogenome sequencing, to provide reference sequences for OTU assignment and mito-metagenome assembly respectively.