To investigate the oral microflora of patients with oesophageal squamous cell carcinoma (ESCC), saliva samples were collected from 20 patients with ESCC and 21 healthy controls. The V3-V4 region of 16S rDNA was amplified and sequenced by the Illumina MiSeq high-throughput sequencing platform. The final sequences were used for OTU analysis. Alpha and beta diversity analysis showed that the bacterial diversity and richness of the ESCC group were lower than those of the control group, while the variability of the ESCC group was higher than that of the control group. According to the Metastats difference analysis and LEfSe analysis, the high risk of ESCC may be related to Actinomyces and Atopobium, while the healthy control group is closely related to Fusobacterium and Porphyromonas (the analysis was performed at the genus level). The establishment of the relationship between oral microbiota and risk of ESCC may lead to significant advances in understanding the aetiology of cancer and may open a new research paradigm for cancer prevention.