One of the main advantages of the application of shot gun sequencing metagenomics to environmental DNA samples is the study of the whole biological community without taxonomic constrains. This can be especially powerful when combined with environmental DNA (eDNA) samples from freshwater systems since they are not only composed by molecules from local aquatic organism but also from organism living in the surrounding landscape. However, the technical limitations of such methodology to study multicellular eukaryotic organisms such as animals and plants have yet not been fully studied. In the present project we test these by studying the freshwater communities from the River Ave in Northern Portugal. Seven samples were collected and used to produce two sequencing datasets. We discuss possible new levels of information’s that metagenomics can provide as well as some of its limitations. This is done by playing a particular focus on the freshwater fish community since it is better described than other groups. We produced two types of datasets. First, a classical metabarcoding data using the MiFish primers targeting the mitochondrial 12S region from fish species. For these we also constructed a reference database of the 12S mitochondrial region. Second, an Illumina MiSeq shot gun sequencing dataset for the same sample set.