| Summary: | Two novel strains of <i>Rummeliibacillus</i> sp. and <i>Microbacterium</i> sp. were identified from the intestine of olive flounder (<i>Paralichthys olivaceus</i>) and characterized in vitro as potential probiotics. Feeds without probiotic and with a 50:50 mixture of these two strains (1 × 10<sup>8</sup> CFU/g feed) were denoted as the control and Pro diets, respectively. Three randomly selected tanks (20 flounders/tank, ~11.4 g each) were used for each diet replication. After 8 weeks of feeding, the growth and feed utilization of the flounder in the Pro group improved (<i>p</i> < 0.05) compared to the control. Among four immune parameters, only myeloperoxidase activity was elevated in the Pro group. Serum biochemistry, intestinal microbial richness (Chao1), and diversity (Shannon index) remained unchanged (<i>p</i> ≥ 0.05), but phylogenetic diversity was enriched in the Pro fish intestine. Significantly lower Firmicutes and higher Proteobacteria were found in the Pro diet; the genus abundance in the control and Pro was as follows: <i>Staphylococcus</i> > <i>Lactobacillus</i> > <i>Corynebacterium</i> and <i>Lactobacillus</i> > <i>Staphylococcus</i> > <i>Corynebacterium</i>, respectively. Microbial linear discriminant scores and a cladogram analysis showed significant modulation. Therefore, the combination of two host-associated probiotics improved the growth and intestinal microbial population of flounder and could be supplemented in the Korean flounder industry.
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