Estuaries provide critical ecosystem services, and yet are increasingly under threat from urbanization. Non-invasive approaches to monitor biodiversity resident to or migrating through estuaries is critical to evaluate the holistic health of these ecosystems, often based entirely on water quality. In this study we compared tree of life metabarcoding (ToL-metabarcoding) biodiversity detections with measurements of physico-chemical variables (chlorophyll a, turbidity, total nitrogen, total phosphorous, dissolved oxygen) at eight sites of varying degrees of water quality in the Gold Coast Broadwater Estuary (Queensland, Australia). These sites were ranked according to an adapted Water Quality Index (WQI) score. Here, we detected 787 unique taxa, adding 137 new biodiversity records to the region, mostly micro-organisms such as bacteria, ciliates, diatoms, dinoflagellates, and cryptomonads. Sites with the lowest WQI were characterised by higher turbidity, lower dissolved oxygen, as well as higher total nitrogen and phosphorous, which correlated with an increased diversity of bacteria, ciliates, and green algae. Similarly, the composition of taxa was significantly different between sites with variable WQI values for most taxa but was less apparent for larger vertebrate groups. These findings suggest that rapid ToL-metabarcoding biodiversity detections, particularly for lower order taxonomic groups, can serve as valuable indicators of flora and fauna across the tree of life associated with dynamically shifting estuarine health along urbanized coastlines.