Objective To analyze the characteristics of the intestinal microflora in patients with breast fibroadenoma using 16S ribosomal RNA (rRNA) high-throughput sequencing. Methods Fecal samples from 20 patients with breast fibroadenoma and 36 healthy subjects were randomly collected and analyzed using high-throughput sequencing technology for 16S rRNA V4 region sequencing, and the alpha diversity (Chao index, Shannon index) was calculated using Mothur (v.1.39.5) software. Beta diversity was analyzed using QIIME (v1.80). SPSS software (version 23.0) and the t-test of two independent samples were used to analyze differences in the abundance of bacteria between the two groups. Results Compared with that in the healthy control group, the α diversity of the intestinal microflora in breast fibroadenoma patients increased, but the difference was not statistically significant (P > 0.05). At the phylum level, significant differences were observed between the two groups. The abundance of Firmicutes was higher in the breast fibroadenoma group (P < 0.05), whereas the abundance of Synergistetes was higher in the healthy control group (P < 0.005). A total of five bacterial genera showed significant differences between the two groups: the breast fibroadenoma group showed higher levels of Bautia (P < 0.005), Coprococcus (P < 0.005), Roseburia (P < 0.05), and Ruminococcus (P < 0.005), whereas Sutterella was more abundant in the healthy control group than in the breast fibroadenoma group (P < 0.05). Conclusion The diversity and abundance of the intestinal flora in patients with breast fibroadenoma are significantly different from those in healthy subjects, suggesting that an imbalance in the intestinal flora is correlated with the occurrence of breast fibroadenoma.