The Osumi Strait and its neighboring waters are major spawning grounds for commercially important small pelagic and coastal fish in the Pacific coastal area of Japan. Mesopelagic fish larvae are also abundant here, co-occurring with the larvae of commercial species. However, it is unknown whether these fish groups compete for prey. We examined the diet of four commercial fish groups (Engraulis japonicus, Trachurus japonicus, Triglidae spp., and Sebastiscus spp.) and two mesopelagic species (Sigmops gracilis, Myctophum asperum) during their larval stages to determine whether they had the same prey source. Morphological analysis and DNA metabarcoding of gut contents showed that the main prey species of all six fish groups were calanoid copepods. However, results from metabarcoding differed greatly from morphological analysis, showing that appendicularians were abundant in the guts of S. gracilis, M. asperum, and Sebastiscus spp. This may be because the methods differ in how they determine prey composition, with morphological analysis relying on counts of identifiable prey parts and metabarcoding being only a semi-quantitative method. In addition, appendicularians might be underestimated by metabarcoding analysis due to variation in genome size, copy number of target genes, and primer mismatches. Stable isotope analysis supported the importance of appendicularians as prey for S. gracilis, M. asperum, and Sebastiscus spp. Our results indicate that the two mesopelagic species and Sebastiscus spp. may compete for prey, but E. japonicus, T. japonicus, and Triglidae spp. occupy niches different from those of the two mesopelagic species. Our results imply that the trophic pathway via appendicularians may support the feeding of dominant mesopelagic species and enable coexistence with commercial species in the study area.