Low-level laser therapy (LLLT) is used in the treatment of chemoradiotherapy- or radiotherapy-induced oropharyngeal mucositis (ORM). In head and neck cancer, tumor cells may lie in the LLLT irradiation field, and LLLT might promote tumor progression. We therefore investigated the effect of LLLT on proliferation, cell cycle distribution, and apoptosis in a human oral carcinoma cell line (SCC-25), non-malignant epithelial cells (BEAS-2B), and fibroblasts in vitro. The cell lines were subjected to LLLT on three consecutive days for 15 min. Cell proliferation was assessed using the MTT assay, cell cycle distribution by flow cytometry and propidium-iodide DNA staining, and apoptosis using an Annexin V-FITC assay. Controls were sham-treated, but not exposed to the laser treatment. LLLT treatment resulted in increased fibroblast proliferation (p < 0.001), whereas decreased cell proliferation was observed after LLLT treatment of BEAS-2B (p = 0.003) and SCC-25 cells (p < 0.001). In SCC-25 cells, an increased percentage of S-phase cells and decreased percentage of G1-phase cells were observed (p < 0.001). Moreover, a proapoptotic effect of LLLT was observed in SCC-25 cells (p = 0.02). LLLT did not exhibit a tumor-promoting effect in this in vitro study.