Coral reefs are biodiversity hotspots, places of high endemicity and provide essential services to billions of people globally. With increasing threats to these reefs worldwide, there is a need to implement faster, more efficient ways to monitor spatial and temporal patterns of biodiversity. Environmental DNA (eDNA) metabarcoding offers a promising tool to address this issue, as it has revolutionized our ability to monitor biodiversity from complex environmental samples such as seawater. However, the technique’s capacity to resolve fine scale shifts in community composition between habitats across the reefscape is not fully explored. Here, we applied eDNA metabarcoding using the universal 18S eukaryote assay to explore differences in community profiles between samples collected from the lagoon and reef slope zones across more than 200 km of the Ningaloo Reef in Western Australia. We recovered 2,061 Amplicon Sequence Variants (ASVs) that comprised of 401 taxa spanning 14 different metazoan phyla including the algae Rhodophyta and Chlorophyta. Our results revealed strong clustering of samples by habitat type across the length of the reef. Community dissimilarity (beta diversity) between samples collected from the reef slope and lagoon habitats was high and was driven largely by a strong rate of spatial turnover, indicating a distinct set of taxa representing each reef zone community. Despite high connectivity due to regular flushing of the lagoon environment, our results demonstrate that metabarcoding of seawater eDNA from different habitats can resolve fine scale community structure. By generating multi-trophic biodiversity data, our study also provided a baseline for Ningaloo Reef from which future changes can be assessed.