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Background: In response to the poor prognosis of oral squamous cell carcinoma (OSCC), the most frequent cancer in the oral cavity, efforts have been made to create more effective treatment methods based on natural products. Lichens are a source of active pharmacologic compounds, which have lately been discovered to be helpful in cancer treatment. Current research seeks to evaluate the impact of Ramalina sinensis (R. sinensis) lichen on the cell viability and apoptosis of OSCC cell lines, considering lichens’ anti-inflammatory and anticancer capabilities. Methods: R. sinensis was selected for investigation of its effects on human oral squamous cell carcinoma cell line (KB cell line, NCBI Code: C152) and acetone and effect of methanol extract of R. sinensison OSCC cell line was investigated. The chemical composition and metabolic profiling of R. sinensis were investigated. Viability was assessed by MTT assay analysis, and apoptotic cells were measured using flow cytometry analysis. Scratch assay was used to assess cell migration. Results: The chemical composition of R. sinensis was investigated using LC-ESI-MS/MS, and 33 unique compounds in acetone and methanol extract of R. sinensis were detected. The proliferation of KB cells was gradually but significantly inhibited by 6.25, 12.5, 25, 50, 100, and 200 μg/mL in a time-dependent manner. There was a statistically significant difference between acetone and methanol extract in the inhibition of cell proliferation. Flow cytometry results indicate an increase in apoptosis of OSCC cells by acetone extract. R. sinensis significantly inhibited the migration and invasion of OSCC cells in a concentration-dependent manner. Conclusion: The results of the present study showed the positive effects of R. sinensis extract on the apoptosis of OSCC cells and the anticancer effects of R. sinensisextracts.