Marine biofouling remains an unsolved problem with a serious economic impact on several marine associated industries and constitutes a major vector for the spread of non-indigenous species (NIS). the implementation of biofouling monitoring programs allows for better fouling management and also for the early identification of NIS. However, few monitoring studies have used recent methods, such as metabarcoding, that can significantly enhance the detection of those species. Here, we employed monthly monitoring of biofouling growth on stainless steel plates in the Atlantic port of Leixões (Northern Portugal), over one year to test the effect of commercial anti-corrosion paint in the communities. Fouling organisms were identified by combining morpho-taxonomy identification with community DNA metabarcoding using multiple markers (16S rRNA, 18S rRNA, 23S rRNA, and COI genes). The dominant colonizers found at this location were hard foulers, namely barnacles and mussels, while other groups of organisms such as cnidarians, bryozoans, and ascidians were also abundant. Regarding the temporal dynamics of the fouling communities, there was a progressive increase in the colonization of cyanobacteria, green algae, and red algae during the sampled period with the replacement of less abundant groups. The tested anticorrosion paint demonstrated to have a significant prevention effect against the biofouling community resulting in a biomass reduction. Our study also reports, for the first time, 29 NIS in this port, substantiating the need for the implementation of recurring biofouling monitoring programs in ports and harbours. Marine biofouling organisms colonize immersed surfaces and form communities on diverse biogenic habitats 1,2. This colonization process is most intense in coastal or shallow waters, where species diversity, temperatures, nutrient levels, and availability of submerged substrata are usually higher compared to offshore areas 3. Biofouling represents a major economic issue for maritime industries and it raises important environmental concerns due to increased drag, exhaust emissions, and operational costs 4,5 while creating also the potential for invasion by non-indigenous species (NIS) 6-8. Stainless steel surfaces are frequently used as a material in vessels, port installation surfaces, cooling water circuits, ships or related equipment, and are known to be extensively colonized by fouling organisms when submerged in water 9,10. Biofouling monitoring programs are therefore essential to help in the understanding and mitigation of such negative impacts. Until recently, the diversity of biofouling organisms has been mainly assessed through morphological identification 11. However, the implementation of methods that allow for earlier and more comprehensive detection of the micro-and macro-fouler colonizers is desirable 12,13. Metabarcoding approaches are useful to validate species identification based on morphological traits, with the possibility of "sight-unseen" detection of target species 14,15 , and detecting of...