This study aimed to characterize salivary microbiota in patients with oral cancer using 16S rRNA amplicon sequencing. DNA was extracted from saliva samples of 23 patients with oral cancer and 95 age-matched controls. A metagenomic analysis was performed using 16S rRNA amplicon sequencing. Patients with oral cancer exhibited lower α-diversity, as indicated by the Chao-1 index, compared to the control group, and significant differences in β-diversity were observed between the two groups. At the genus level, 25 bacterial species such as Lautropia, Megasphaera, Lactobacillus, Kingella, Gemella, Staphylococcus, and Propionibacterium were identified in patients with oral cancer, with more than half being Gram-positive facultative anaerobes or anaerobes. The reduced bacterial diversity in saliva of patients with oral cancer suggests dysbiosis during oral carcinogenesis may contribute to changes in bacterial distribution within the oral cavity.