Rationale Approximately 20-40% of patients with cancer will experience brain metastasis (BM), which has a great impact on the quality of life and survival rates of patients. Whole brain radiotherapy (WBRT) is an effective method for the treatment of BM. However, it cannot be ignored that WBRT might induce a series of neuropsychiatric side effects, including cognitive dysfunction (CD). Accumulating evidence shows that the gut microbiota and the gut-microbiota-brain axis may play a vital role in the pathogenesis of CD. Objective and methods We adopted WBRT to mimic CD after a hierarchical cluster analysis of the Morris water maze test (MWMT) results. In addition, we observed the effects of antibiotics and prebiotics on WBRT-induced CD. Variations were revealed via the 16S rRNA sequencing analysis at different levels. Results The 16S rRNA sequencing analysis revealed an altered composition of gut microbiota between CD and non-CD phenotypes. Furthermore, we observed a decrease in the levels of Phylum-Bacteroidete, Class-Bacteroidia, and Order-Bacteroidales in the CD group and an increase in the Genus-Allobaculum level after WBRT. Pretreatment with antibiotics caused a significant decrease in the level of Phylum-TM7 01, whereas an increase in the levels of Class-Gammaproteobacteria, Order-Enterobacteriales, and Species-Escherichia coli. After pretreatment with probiotics, the levels of Phylum-Cyanobacteria, Class-4C0d-2, and Order-YS2 were decreased, while the levels of Family-Bacteroidaceae, Genus-Bacteroides, and Species-Parabacteroides distasonis were increased. Conclusions WBRT-induced CD might be highly related to abnormal composition of gut microbiota. Strategies improving the composition of the gut microbiota may provide beneficial effects on CD in individuals exposed to WBRT.