The global biodiversity crisis driven by anthropogenic pressures significantly threatens marine ecosystems functioning. The rate of climate change and the impacts of anthropogenic pressures outpacing the capabilities of our observation tools, stresses the need to develop new methods to assess these rapid modifications. Environmental DNA (eDNA; DNA traces released by organisms) metabarcoding has emerged as a non-invasive method that has been widely developed over the last decade. Thanks to a large spatio-temporal coverage, high detection of rare species and its time and cost effectiveness, eDNA metabarcoding represents a promising biomonitoring tool. However, capturing fish diversity using eDNA requires a high-quality genetic reference database, which we are currently still lacking. For the South European Atlantic shelf area, we estimated that only 41% of the fish species present were recorded in the available eDNA reference databases. Improving reference databases can notably rely on opportunistic sampling enabling the reporting of sequences for new species. Therefore, the data provided here consists of barcoding 95 species of ray-finned and cartilaginous fishes over the 12S mitochondrial DNA gene. We generated 168 12S barcodes from fishes that were sampled in the Bay of Biscay (Northeast Atlantic, France) between 2017 and 2019. We also provided the “Teleo” barcode associated with a specific 12S region for each individual. In addition to the sequences, we provided for each individual the taxonomy, the details associated with the barcode (Genbank accession number, chromatograms), a photograph, as well as 5 ecomorphological measures and 11 life-history traits. These traits document several functions such as dispersion, diet, habitat use, and position in the food web. Furthermore, we provided the metadata of each sampling site (date, station, sampling hour, gear, latitude, longitude, depth) and environmental variables measured in situ (conductivity, salinity, water temperature, water density, air temperature). This data set is highly valuable to improve the Northeast Atlantic eDNA genetic database, thus helping to better understand the effects of environmental forcing in the Bay of Biscay, a transition zone housing mixed assemblages of boreal, temperate and subtropical fish species susceptible to display variability in functional traits to adapt to changing conditions.