Macroalgae are key primary producers in North Atlantic and Arctic coastal ecosystems, and tracing their fate and distribution is vital to improve our understanding of their ecological role and provision of ecosystem services. Recent advances from environmental DNA (eDNA) have added a new capacity to fingerprint and trace macroalgae. However, further development of resources for amplifying and identifying macroalgal eDNA are much needed. Here, we examined the performance in terms of resolution and specificity of two 18S primers (18S-V7 and 18S-V9) recently applied in identifying macroalgae from eDNA. We also built a local barcode database for primer 18S-V7 with 31 widespread Arctic and North Atlantic macroalgal species to complement the existing DNA databases. Furthermore, we applied metabarcoding of eDNA to identify macroalgae in Arctic marine sediments (Disko Bay, W. Greenland) and evaluated the contributions from our local barcode database. We identified macroalgal DNA from 19 families across 11 orders in surface (0–1 cm, with both primers) and sub-surface (5–10 cm, with 18S-V7 primer) sediments. The barcode database developed here with the 18S-V7 primer improved the identification of unique families, from 16 to 19 families, thereby strengthening the taxonomic assignment possible relative to pre-existing barcode reference sequences. Overall, this study demonstrates the feasibility of eDNA to resolve contributions of macroalgae in Arctic marine sediments, and enhances the fingerprinting resolution. We thereby document a novel pathway to answer key questions on the ecological role and fate of macroalgae in the Arctic.