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Large-scale data synthesis is a foundation pillar of modern macroecology, necessary for transitioning fragmented regional data into scalable biosecurity frameworks. This study presents the outcomes of a coordinated nationwide effort to analyze marine Non-Indigenous Species (NIS) records across mainland Portugal, the Azores and Madeira archipelagos using a dual-framework design. Our goals were to update the morphologically confirmed NIS inventory, identify primary introduction pathways, examine temporal and spatial trends, and evaluate genetic database coverage. We delivered an updated checklist comprising 231 morphologically confirmed marine NIS, including five new regional records. Spatial analysis reveals distinct biogeographical divergence: mainland NIS predominantly originate from the temperate North Pacific, probably due to shipping and aquaculture vectors, whereas archipelago NIS originate from the North Atlantic. This pronounced regional variability in pathway dynamics underscores the need for spatially specific management rather than uniform national policies. Also, a systematic genetic gap analysis showed that 71% of these species are cataloged in global repositories, whereas only 12.5% feature locally sourced sequences. To bridge this gap, the second framework introduces a precautionary molecular watchlist of 22 species detected exclusively via DNA-based methods awaiting morphological confirmation. Ultimately, this dual-framework structure demonstrates how integrating taxonomy with provisional molecular data provides a reliable, data-driven baseline, while moving beyond a localized dataset update to offer a scalable reference frame capable of informing regional and global marine biosecurity frameworks.