Main prognostic factors of anal squamous cell carcinoma (ASCC) are tumor size, differentiation, lymph node involvement, and male gender. However, they are insufficient to predict relapses after exclusive radiotherapy (RT) or chemoradiotherapy (CRT). Fusobacterium nucleatum has been associated with poor prognosis in several digestive cancers. In this study, we assessed the association between intratumoral F. nucleatum load and clinico-pathological features, relapse, and survival in patients with ASCC who underwent abdominoperineal resection (APR) after RT/CRT. We retrospectively analyzed surgical samples from a cohort of 166 patients with ASCC who underwent APR. F. nucleatum 16S rRNA gene sequences were quantified using real-time quantitative PCR. We associated F. nucleatum load with classical clinicopathological features, overall survival (OS), disease-free survival (DFS), and metastasis-free survival (MFS) using Cox regression univariate and multivariate analyses. Tumors harboring high loads of F. nucleatum (highest tercile) showed longer OS and DFS (median: not reached vs. 50.1 months, p = 0.01, and median: not reached vs. 18.3 months, p = 0.007, respectively). High F. nucleatum load was a predictor of longer OS (HR = 0.55, p = 0.04) and DFS (HR = 0.50, p = 0.02) in multivariate analysis. High F. nucleatum load is an independent favorable prognostic factor in patients with ASCC who underwent APR.