Small volumes of water containing environmental DNA (eDNA) are increasingly combined with metabarcoding to generate biodiversity data for specific fractions of marine flora and fauna. To date, however, few studies have utilized this technique to assess how well it captures seasonal patterns in coral reef communities or how environmental and methodological factors influence eDNA detections. In our study, we used three eDNA metabarcoding assays primarily targeting bony fish and elasmobranchs, as well as cnidarians and sponges (Cnidaria/Porifera) combined with monthly seawater sampling to (1) investigate temporal variation in taxonomic detections and (2) statistically test the potential effect of season, sea surface temperature, timing of spawning (using moon phase as a proxy), and sample preservation on taxon detection across a 12-month period in a model coral reef system (Big Vicki’s Reef, Lizard Island, Great Barrier Reef, Australia). Species-level fish and genus-level scleractinian coral detections from standardized visual surveys conducted at the same coral reef, in addition to a curated list of all known fishes recorded from the more expansive coral reef system across 46 years, were used to validate eDNA detections. Our eDNA dataset indicated that the number of taxa detected were consistently highest in September for fish, and in February followed by September for Cnidaria/Porifera. Conversely, detections were lowest in June and July for all taxa. Some, but not all, of the environmental and methodological variables explained the observed temporal pattern in biological communities or systematic changes in the number of taxa, and in some cases, this effect was taxon dependent. Our study also highlights the significance of timing in eDNA biodiversity surveys conducted on tropical coral reefs in the Southern Hemisphere. To obtain the most meaningful estimates of site diversity, we recommend focusing sampling efforts between early spring and early autumn. Alternatively, allocating an entire year to sampling would better capture seasonal variation and provide more comprehensive insights into coral reef biodiversity.