Non-indigenous species (NIS) pose significant threats to coastal ecosystems, causing ecological imbalances and biodiversity loss. Effective monitoring and management strategies are essential to mitigate these impacts, particularly in marinas and ports, which are crucial entry points for NIS due to shipping. This study assessed the spatial distribution of marine invertebrate fauna, particularly NIS, in 10 recreational marinas in Portugal (6 on the mainland and 2 each in Madeira and Azores) using DNA metabarcoding. Samples were collected from hard substrates, water and zooplankton in a single time-point survey in June and July 2020. Using the mitochondrial cytochrome c oxidase subunit I (COI) and the small subunit 18S rRNA gene (18S) markers, a total of 645 species, including 38 NIS, were detected. Only 5% of species and 5% of NIS were common to all marinas. The highest species richness was observed in the mainland North marinas, while Madeira exhibited the lowest richness. Ponta Delgada (Azores) recorded the highest number of NIS (15), whereas Madeira’s marinas recorded the lowest (7 and 9). Species richness and taxonomic composition varied with sample type. A Principal Coordinate Analysis (PCoA) revealed three distinct groups: (1) North and Aveiro (mainland); (2) Lisbon (mainland); and (3) Madeira and the Azores. A PERMANOVA supported the theory that both sample type and marina significantly influenced species composition [P(perm) = 0.001]. A comparison of the 38 NIS detected in this study, with the 2020 national NIS lists, revealed regional differences and spotted species that were not previously considered NIS but are now recognized as such: eight NIS in mainland Portugal, two in Madeira and two in Azores. This study provides valuable insights into the distribution of NIS and marine invertebrate fauna in Portuguese recreational marinas, while highlighting the efficacy of DNA metabarcoding for assessing spatial variability in invertebrate communities and supporting NIS surveillance programs.