Metabolites derived from marine symbiotic microorganisms have great potential as lead compounds for the discovery of novel marine drugs. Euphausia superba Dana, which lives in the Antarctic Ocean, is regarded as a new source of marine microbial natural products. However, no studies have examined the biodiversity of the symbiotic intestinal microbiome of E. superba. To address this issue, the species diversity and abundance of the gut microbiome of E. superba Dana from the Rose Sea of the Antarctic Ocean were analyzed by culture-independent high-throughput sequencing and pure culture methods. A comparison with gene databases revealed that the microbiome contained 61 known microbial species and a plethora of uncultivable microorganisms. Additionally, 7% of the species in the microbiome were currently unknown. The microbes belonged to 56 genera, eight of which, including Arthrobacter, Bacillus, Candidatus, Lactococcus, Lysinibacillus, Leuconostoc, Solibacillus, and Vibrio, were dominant, as were Vibrionaceae spp. Moreover, 81 microbial strains were isolated by the pure culture method, and they belonged to 36 genera, including Mobilicoccus, Rhodococcus, Arthrobacter, and Microbacterium. The results obtained by two different methods demonstrate the richness of the microbial biodiversity of the gut microbiome of E. superba, and it also suggests that they have good potential for the discovery of novel marine microbial species. Metabolites derived from marine symbiotic microorganisms have great potential as lead compounds for the discovery of novel marine drugs. Euphausia superba Dana, which lives in the Antarctic Ocean, is considered as a new source of marine microbial natural products. However, no studies have examined the biodiversity of the symbiotic intestinal microbiome of E. superba. To address this issue, the species diversity and abundance of the gut microbiome of E. superba Dana from the Rose Sea of ​​the Antarctic Ocean were analyzed by culture-independent high-throughput sequencing and pure culture methods. A comparison with gene databases revealed that the microbiome contained 61 known microbial species and a plethora of uncultivable microorganisms. Additionally, 7% of the species in the microbiome were currently unknown. The microbes belonged to 56 genera, eight of which, including Arthrobacter, Bacillus, Candidatus, Lactococcus, Lysinibacillus, Leuconostoc, Solibacillus, and Vibrio, were dominant, as were Vibrionaceae spp. Moreover, 81 microbial strains were isolated by the pure culture method, and they belonged to 36 genera, including Mobilicoccus, Rhodococcus, Arthrobacter, and Microbacterium. The results obtained by two different methods demonstrate the richness of the microbial biodiversity the gut microbiome of E. superba, and it also suggests that they have good potential for the discovery of novel marine microbial species.