OBJECTIVESWe compared the oral bacteriome and mycobiome of patients with oral submucosal fibrosis (OSMF) and oral squamous cell carcinoma (OSCC) who consumed tobacco/betel quid with those of healthy individuals.METHODSTargeted amplicon sequencing coupled with bioinformatics analysis was performed to assess the bacterial and fungal communities in the oral cavity.RESULTSDistinct variations in the microbial communities were observed among the healthy, OSMF, and OSCC samples. Firmicutes was dominant at the phylum level: 21.71%, 21.73%, and 24.56% in the healthy control, OSMF, and OSCC samples, respectively. Lactobacillus (9.6%, 9.04%, and 8.9%) and Streptococcus (7.3%, 7.55%, and 6.1%) showed minimal variation among the healthy, OSMF, and OSCC samples, respectively, at the genus level. Rothia (2.73%) and Veillonella (1.52%) were prominent in those with OSMF, and Gemella (2.2%) and Fusobacterium (1.5%) were prominent in the OSCC samples. The oral mycobiome was dominated by Ascomycota and Basidiomycota in all samples, with Ascomycota accounting for 18.95%, 16.05%, and 15.87%, and Basidiomycota for 13.55%, 15.79%, and 15.96% in the OSCC, OSMF, and healthy samples, respectively. PICRUSt2 analysis revealed the key metabolic pathways linked to disease and xenobiotic degradation, related to the harmful compounds from tobacco, highlighting the clinical relevance of tobacco and betel quid associated microbial communities as well as the differences between OSCC and OSMF.CONCLUSIONSMicrobial signatures associated with OSCC and OSMF could serve as biomarkers for early cancer detection. These findings highlight how tobacco and betel quid consumption promote cancer and alter the oral microbome.