Background <p>The <i>Bifidobacterium</i> genus is a prominent bacterial population in the gastrointestinal tract. Previous findings suggest that <i>Bifidobacterium</i> is linked to tumor suppression in mouse models of melanoma. Additionally, when combined with the programmed death-ligand 1 (PD-L1) antibody, it can enhance anti-tumor treatment by increasing tumor-specific T-cell responses and promoting infiltration of antigen-specific T cells into tumors. However, there is a lack of studies on <i>Bifidobacterium</i> in esophageal squamous cell carcinoma (ESCC). This study aimed to investigate the potential impact of <i>Bifidobacterium</i> on this cancer type.</p> Methods <p>We examined 213 samples from ESCC patients who underwent tumor resection. The presence of <i>Bifidobacterium</i> was confirmed using quantitative polymerase chain reaction and fluorescent in situ hybridization (FISH). Patient overall survival (OS) was analyzed with <i>Bifidobacterium</i> positivity. Tumor-infiltrating lymphocytes (TILs) were evaluated via hematoxylin and eosin stains, and immunohistochemistry was used to assess programmed death-1 (PD-1), PD-L1, cluster of differentiation 8 (CD8), and forkhead box P3 (FOXP3) expression. Nutritional status was evaluated via computed tomography scans.</p> Results <p><i>Bifidobacterium</i> positivity showed no correlation with patient OS or TIL levels; however, <i>Bifidobacterium</i> positivity in normal tissue was associated with lower FOXP3 levels, suggesting a potential role in upregulating anti-tumor immune responses. Patients with <i>Bifidobacterium</i> present in peritumor normal tissue exhibited better skeletal muscle area and volume. Conversely, <i>Bifidobacterium</i> positivity in tumor tissue was associated with poorer prognostic nutrition index values, likely due to decreased albumin levels.</p> Conclusion <p><i>Bifidobacterium</i> can induce the upregulated anti-tumor immune response and is more prevalent in cases with good nutritional status.</p>