Mesophotic coral ecosystems (MCEs) are light-dependent communities occurring at depths of 30–150 m. They have been suggested to serve as refuge against thermal stress during heat waves for some coral species. Recent studies on MCEs have revealed a high diversity of communities, some unique, and that these ecosystems are far from being immune to anthropogenic threats. However, the depths at which these ecosystems are found make their exploration and study challenging. Consequently, most suitable environments for MCEs remain unexplored. To facilitate the detection and characterization of MCEs, we improved the methodology for mesophotic scleractinian survey by environmental DNA (eDNA) metabarcoding analysis using seawater collected by underwater mini-Remote Operated Vehicle (mini-ROV). We tested this improved approach at upper mesophotic sites in Okinawa, Japan, with different corals dominating the communities (i.e., Alveopora-dominated, Seriatopora-dominated, and Acropora-dominated communities). Despite the proximity of the different sites, our eDNA metabarcoding analyses detected the dominant coral genera specific to each site. In addition, this study detected numerous other genera present at these sites, including Acropora, Pachyseris, Galaxea, Lobophyllia, Montipora, Pocillopora, Porites, and others. Therefore, this study might support a new technical gate for comprehensive survey of MCEs using eDNA samples collected by underwater mini-ROV, although further technical improvement is required for quantitative estimation.