We sampled seawater from nine mesophotic reef sites in the Pinnacles Trend and DeSoto Rim areas in the northern Gulf of America in October 2022 to complement and inform restoration investments for coral communities. Four primer sets were used to amplify environmental DNA (eDNA) from seawater, targeting metazoans, eukaryotic plankton and protists, corals, and fishes. Across all markers, four Kingdoms were detected including Animalia, Chromista, Plantae, and Protista based on 11,793 amplicon sequence variants (ASVs). Within Kingdom Animalia, 11 Phyla containing 33 Classes were detected from the surface to the seafloor. Corals and fishes, which are of particular interest, were detected at all sites and in congruence with previous video sightings for the area.In some cases eDNA analyses were able to identify fish and coral to a lower taxonomic identification than was possible through video analyses. This study provides evidence that the addition of an eDNA component to habitat characterization and biodiversity assessments can provide essential information about ecosystem dynamics from the surface, through the water column, to the benthic community.