Coastal areas host a major part of marine biodiversity but are seriously threatened by ever‐increasing human pressures. Transforming natural coastlines into urban seascapes through habitat artificialization may result in loss of biodiversity and key ecosystem functions. Yet, the extent to which seaports differ from nearby natural habitats and marine reserves across the whole Tree of Life is still unknown. This study aimed to assess the level of α and β‐diversity between seaports and reserves, and whether these biodiversity patterns are conserved across taxa and evolutionary lineages. For that, we used environmental DNA (eDNA) metabarcoding to survey six seaports on the French Mediterranean coast and four strictly no‐take marine reserves nearby. By targeting four different groups—prokaryotes, eukaryotes, metazoans and fish—with appropriate markers, we provide a holistic view of biodiversity on contrasted habitats. In the absence of comprehensive reference databases, we used bioinformatic pipelines to gather similar sequences into molecular operational taxonomic units (MOTUs). In contrast to our expectations, we obtained no difference in MOTU richness (α‐diversity) between habitats except for prokaryotes and threatened fishes with higher diversity in reserves than in seaports. However, we observed a marked dissimilarity (β‐diversity) between seaports and reserves for all taxa. Surprisingly, this biodiversity signature of seaports was preserved across the Tree of Life, up to the order. This result reveals that seaports and nearby marine reserves share few taxa and evolutionary lineages along urbanized coasts and suggests major differences in terms of ecosystem functioning between both habitats.