Successful feeding on preferred prey could be important for the survival of fish larvae. However, high taxonomic resolution of prey types from damaged gut contents is difficult to achieve through morphological classification, especially for fragile organisms or immature stages. Here, we applied 18S metabarcoding diet analysis to total 64 early post-larvae (< 10 mm) of Japanese sardine Sardinops melanostictus and Pacific round herring Etrumeus teres, which were collected from Tosa Bay (Japan) during their main spawning periods. There were no clear differences in the diets of the co-existing fish species, and copepods comprised the majority of the prey items. Among the copepods, the OTU derived from the large copepod Calanus sinicus was most consumed, followed by the OTU identified as small Paracalanus sp. Considering body size of copepods, the fish larvae actively consumed nauplii of C. sinicus, suggesting that both food availability and developmental stages of copepods determined prey preference. Additionally, we applied the same technique to 40 adult female C. sinicus. The majority of prey items were small crustaceans and diatoms, taxa that are dominant in the environment and have been previously reported as important prey items of Calanus. Aplanochytrids (Labyrinthulea) were detected for the first time as a major prey of C. sinicus, and high proportions of unclassified eukaryote material were also observed. These results suggest that further investigation into novel predator–prey relationships is recommended to understand the complex food web structures and population dynamics of commercially important fish species in marine ecosystems.