Safeguarding marine ecosystems is essential for maintaining ecosystem function and biodiversity, but effective monitoring of marine habitats can be logistically challenging, costly, and difficult to regularly implement. Environmental DNA-based biomonitoring is a rapidly growing tool that is non-destructive, cost-effective, and reliable. However, discrepancies in eDNA sampling protocols and methodology persist, which can greatly impact the interpretations of biomonitoring results, particularly across highly diverse ecosystems with historically elevated biodiversity. The South African coastal system is a unique and highly diverse ecoregion consisting of two ocean boundary currents creating one of the most diverse biological regions on the planet. Here, we present the first eDNA-based metabarcoding assessment of South African coastal fishes while also providing key management insights into study and sample design. We observed strong ecological associations with fish species richness across the extent of the South African coast, along with weaker associations with seasonality. We detected 466 operational taxonomic units across 112 of the 270 families described previously from the region, with greater species richness on the eastern subtropical coast compared to the western coast, which follows expected species richness patterns. Additionally, we provide evidence that biological replication is necessary to detect intra-site fish diversity and that three biological replicates are sufficient for capturing species diversity dynamics. Our work highlights the value of eDNA biomonitoring across space and time enabling biodiversity characterizations for the management of a gradient of coastal marine environments.