Marine biofouling communities, including biofilms, are composed of many eukaryotes with high taxonomic and functional diversities. However, molecular characterization of eukaryotic diversity of marine biofouling has been barely developed due to the only recent interest in research areas such as marine renewable energies, antifouling technologies, or plastic pollution. The aim of this study was to compare the diversity and taxonomic composition of biofouling through different metabarcoding approaches used to detect the widest range of taxa from samples collected in several contrasted marine environments (French Atlantic and Mediterranean coasts). Thus, we assessed four DNA extraction methods and six primers pairs targeting the 18S rDNA gene (including the V1-V2, V4TAR, V4UNI, V7 and V9 regions) and the COI gene, the latter with two databases (BOLD and MIDORI). In addition the influence of primers selection was analyzed at three sites to compare geographic variations in eukaryotic diversity. Although none of the extraction methods greatly altered the community diversity or composition. we have observed that eukaryotic biofouling community diversity and structure varied depending on primers pairs, reference databases and sites. 18S rDNA regions allowed the detection of more taxa at the species level, including microeukaryotes, while the COI recovered more ASVs, but with a large proportion that remained taxonomically unassigned probably because BOLD and MIDORI specifically targeted metazoans. Interestingly, the spatial pattern obtained with both COI and 18S rDNA markers were similar showing that spatial selection occurred throughout a wide diversity of eukaryotic taxa. These results encouraged the use of these two complementary markers for future metabarcoding investigations but also highlighted the relevance of completing databases to enhance the identification of biofouling eukaryotes.