Accumulating evidence has identified Fusobacterium as an important pathogenic gut bacterium associated with colorectal cancer. Nevertheless, only limited data exist about the role of this bacterium in locally advanced rectal cancer (LARC). In this study, we quantified Fusobacterium nucleatum in untreated and post-neoadjuvant chemoradiotherapy (nCRT) samples from LARC patients and investigated its association with therapy response and survival. A total of 254 samples from 143 patients with rectal adenocarcinomas were analyzed for the presence and abundance of F. nucleatum using RNA in situ hybridization and digital image analysis. Assay accuracy was determined using infected cell lines and tumor samples with available quantitative PCR data. We studied the impact of F. nucleatum load on pathologic complete response and relapse-free survival. Treatment-induced changes were evaluated in paired pre- and post-nCRT samples (n = 71). Finally, tumor microenvironment changes during nCRT were assessed in paired samples (n = 45) by immune contexture analysis. F. nucleatum tissue levels by RNA in situ hybridization strongly correlated with quantitative PCR (r = 0.804, P < 0.001). F. nucleatum abundance was higher in untreated median, 7.4; 95% confidence interval (3.7–16.2) compared with treated median, 1.6; 95% confidence interval (1.3–2.4) tumors (P <0.001) with 58% (73/126) and 26% (22/85) positive tumors, respectively (P < 0.001). Baseline F. nucleatum levels were not associated with pathologic complete response. F. nucleatum positivity after nCRT, but not baseline status, significantly increased risk of relapse hazard ratio = 7.5, 95% confidence interval (3.0–19.0); P < 0.001. Tumors that turned F. nucleatum-negative after nCRT had a strong increase in CD8+ T cells post-nCRT (P < 0.001), while those that persisted F. nucleatum-positive after nCRT lacked CD8+ T cells induction in post-nCRT samples compared with baseline (P = 0.69). F. nucleatum persistence post-nCRT is associated with high relapse rates in LARC, potentially linked to suppression of immune cytotoxicity. •Biomarkers to predict response or relapse after neoadjuvant chemoradiotherapy (nCRT) remain an unmet clinical need in LARC.•F. nucleatum is significantly enriched in colorectal tumor tissue but its role in rectal cancer is poorly studied.•nCRT significantly reduced F. nucleatum tissue abundance in post-treatment surgical samples.•F. nucleatum persistence after nCRT significantly increased the risk of relapse.•F. nucleatum positivity in post-nCRT associated with lack of immune cytotoxicity activation.