Objective A model for studying oral ulcers induced by betel nut-extract was constructed in rats. Changes in the structure and diversity of oral flora were observed to explore the involvement of oral flora and local inflammatory factors in the pathogenesis of oral ulcers induced by betel nut-extract and to provide theoretical support for the prevention and treatment of oral ulcers in the clinic. Methods Thirty SD rats were randomly divided into normal, model and intervention groups (Guilin watermelon cream, 8 mg/ d for 7 days), with 10 rats/ group. The oral mucosa of rats was subcutaneously injected with 10 g/ mL of betel nut-extract to generate an oral ulcer model. The histomorphological changes were observed, and ulcer area and ulcer scores were assessed. Local oral tissue tumor necrosis factor-α (TNF-α), interleukin (IL)-2 and IL-8 levels were determined. Oral mucosal tissues were sampled for HE staining and analyzed for the structural distribution of oral flora and the diversity of microbial communities using high-throughput sequencing method. Results Compared with rats in the normal group, those in the model group had an increased ulcer area, significantly increased ulcer scores (P< 0. 01), and significantly increased levels of TNF-α, IL-2 and IL-8 in the oral mucosal tissues (P< 0. 01). The amount Streptococcus (P< 0. 05) and Veillonella (P< 0. 001) in the oral saliva of the model group rats was significantly reduced. The model group rats showed oral mucosal epithelial cell hyperplasia or focal necrosis, mucosal lamina propria edema, and hemorrhage accompanied by mass neutrophil and monocyte infiltration. Compared with the model group rats, the intervention group rats had significantly reduced ulcerated area (P< 0. 05, P< 0. 01) and ulcer scores (P< 0. 05). And oral mucosal tissue levels of TNF-α(P< 0. 01), IL-2(P< 0. 05) and IL-8 (P< 0. 05), as well as significantly increased Streptococcus (P< 0. 001) and Veillonella (P< 0. 01) and significantly reduced Staphylococcus (P< 0. 01) in the oral saliva. The degree of lesions in the oral mucosal tissues was significantly improved in the intervention group. Conclusions Betel nut-extract can be used to successfully reproduce a rat model of oral ulcer, and it is speculated that the development of oral ulcers after exposure to betel nut-extract may be related to an imbalance in the oral flora and local tissue inflammatory mediators.