The diversity of the gastrointestinal microbiome is closely associated with human health. In the present study, the gastrointestinal microbiome and tumor-infiltrating lymphocytes (TILs) were compared in patients with breast cancer (BC). A total of 80 patients with BC were divided into three groups based on the expression of TILs, as follows: High expression of TILs (TIL-H), medium expression of TILs (TIL-M) and low expression of TILs (TIL-L). DNA of the gastrointestinal microbiome was determined by Illumina sequencing and taxonomy of 16S ribosomal RNA genes. A (2) test and UniFrac analysis of -diversity were applied to assess the association between clinical characteristics and diversity of the gastrointestinal microbiome. The -diversity distribution was statistically significant (weighted UniFrac, P<0.01; unweighted UniFrac, P<0.01) when comparing the TIL-L and TIL-H groups and when comparing the three groups (TIL-H vs. TIL-M vs. TIL-L). At the genus level, higher abundances of Mycobacterium, Rhodococcus, Catenibacterium, Bulleidia, Anaerofilum, Sneathia, Devosia and TG5, but lower abundances of Methanosphaera and Anaerobiospirillum (P<0.05) were identified in the TIL-L group compared with the TIL-H group. At the species level, the stercoris, barnesiae, coprophilus, flavefaciens and C21_c20 species exhibited a higher abundance in the TIL-L group, whereas producta and komagatae exhibited a greater abundance in the TIL-H group (P<0.05). Collectively, the diversity of the gastrointestinal microbiome was associated with the expression of TILs in patients with BC.