BackgroundDifferent intratumoral microbiotaexist in different tumors and play a crucial function in carcinogenesis. However, whether they impact clinical outcomes in esophageal squamous cell carcinoma (ESCC) and their mechanism remain unclear.Methods16S rDNA amplicon sequencing was performed on surgically resected samples from 98 ESCC patients to analyze intratumoral microbiome abundance and composition. Multiplex fluorescent immunohistochemistry staining was used to profile the phenotypes of immune infiltrates in the tumor microenvironment (TME).ResultsPatients with higher intratumoral Shannon index had significantly worse surgical outcomes. When patients were divided into short-term survivors and long-term survivors based on the median survival time, both intratumoral alpha-diversity and beta-diversity were found to be significantly inconsistent, and the relative abundance ofLactobacillusandLeptotrichiaemerged as the two microorganisms that probably influenced the survival of ESCC patients. OnlyLactobacillusin ESCC was validated to significantly worsen patients’ prognoses and to be positively correlated with the Shannon index. Multivariate analysis revealed that the intratumoral Shannon index, the relative abundance ofLactobacillus, and the pathologic tumor–node–metastasis (pTNM) stage were independently associated with patients’ overall survival. Furthermore, the relative abundance of bothLactobacillusand Shannon index was positively correlated with the proportions of PD-L1+epithelial cells (ECs) and tumor-associated macrophages (TAMs). The Shannon index was negatively correlated with the proportions of natural killer (NK) cells in the TME.ConclusionsA high abundance of intratumoralLactobacillusand bacterial alpha-diversity was associated with the formation of the immunosuppressive TME and predicted poor long-term survival in ESCC patients.