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The fish gut microbiota plays an important role in overall health. However, few reports have described the changes in the composition of gut microbiota following infection with pathogenic bacteria in olive flounder (<i>Paralichthys olivaceus</i>). Here, we reported the changes in the gut microbiota composition of flounder after treatment with each of the three pathogenic bacteria (<i>Edwardsiella tarda</i>, <i>Streptococcus iniae</i>, and <i>Vibrio harveyi</i>). <i>Edwardsiella tarda</i> infection decreased the relative abundance of Verrucomicrobia and increased Proteobacteria abundance at the phylum level of the gut microbiota over time. Similarly, <i>Streptococcus iniae</i> infection reduced the relative abundance of Verrucomicrobia. <i>Vibrio harveyi</i> infection caused a decrease in the relative abundance of Firmicutes and Verrucomicrobia and increased Proteobacteria. At the genus level, infection with all three pathogens increased the relative abundance of <i>Ralstonia</i> and <i>Sphingomonas</i> species. Conversely, this infection decreased the relative abundances of <i>Rubritalea</i>, <i>Saccharimonas</i>, and <i>Bacillus</i> species. Therefore, reducing the abundance of <i>Ralstonia</i> and <i>Sphingomonas</i> and increasing the abundance of <i>Rubritalea</i>, <i>Saccharimonas</i>, and <i>Bacillus</i> in the gut microbiota composition of flounder might help maintain a healthy gut microbiota balance. This research might be useful for future studies on improving the health of flounder through gut microbiota regulation.