Corbicula has often been reported as one of the most invasive freshwater species in the world. It plays an important role in the food chains of brackish water zones in Korea, where it is predominant. However, detailed information on the Corbicula diet is still lacking. The purpose of this study was to identify the potential prey of Corbicula fluminea in the Seomjin River using a DNA metabarcoding approach, as very little is known about its feeding selectivity in natural conditions. A survey was conducted at two study sites (1 and 2) in the Seomjin River in November 2021. The two sites were selected based on increasing salinity gradient. The dominant operational taxonomic unit in the pseudofeces and gut content of C. fluminea was Microcyclops varicans (Copepoda) and Oncorhynchus mykiss (Fish), respectively. The alpha diversity at site 1 was higher than that at site 2. More diverse potential prey species were identified at the site with low salinity (i.e., site 1). The utilization of this method is strongly recommended for determining specific predator–prey relationships in complex estuarine ecosystem.