Urbanization of estuaries drastically changed existing shorelines and bathymetric contours, in turn modifying habitat for marine foundational species that host critical biodiversity. And yet we lack approaches to characterize a significant fraction of the biota that inhabit these ecosystems on time scales that align with rates of urbanization. Environmental DNA (or eDNA) metabarcoding that combines multiple assays targeting a broad range of taxonomic groups can provide a solution, but we need to determine whether the biological communities it detects ally with different habitats in these changing aquatic environments. In this study, we tested whether tree of life metabarcoding (ToL-metabarcoding) data extracted from filtered seawater samples correlated with four known geomorphic habitat zones across a heavily urbanized estuary (Sydney Harbour, Australia). Using this method, we substantially expanded our knowledge on the composition and spatial distribution of marine biodiversity across the tree of life in Sydney Harbour, particularly for organisms where existing records are sparse. Excluding terrestrial DNA inputs, we identified significant effects of both distance from the mouth of Sydney Harbour and geomorphic zone on biological community structure in the ToL-metabarcoding dataset (entire community), as well as in each of the taxonomic subgroups that we considered (fish, macroinvertebrates, algae and aquatic plants, bacteria). This effect appeared to be driven by taxa as a collective versus a few individual taxa, with each taxon explaining no more than 0.62% of the variation between geomorphic zones. Similarly, taxonomic richness was significantly higher within geomorphic zones with large sample sizes, but also decreased by 1% with each additional kilometer from the estuary mouth, a result consistent with a reduction in tidal inputs and available habitat in upper catchments. Based on these results, we suggest that ToL-metabarcoding can be used to benchmark biological monitoring in other urbanized estuaries globally, and in Sydney Harbour at future time points based on detection of bioindicators across the tree of life. We also suggest that robust biotic snapshots can be archived following extensive curation of taxonomic assignments that incorporates ecological affinities, supported by records from relevant and regional biodiversity repositories.