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This study assessed the impact of low-level laser irradiation on the viability and proliferation of human periodontal ligament stem cells (hPDLSCs) cultivated on polylactic acid (PLA) scaffolds. hPDLSCs were obtained, characterized, and grown on the surface of PLA films produced via the solvent casting technique. The study involved two groups: the control group, which was not exposed to radiation, and the laser group, which was irradiated with a diode laser (InGaAIP) with a power of 30 mW, a wavelength of 660 nm, and a single dose of 1 J/cm² emitted continuously. Cell viability was assessed 24 and 48 hours after irradiation using the Alamar blue and Live/Dead assays. Flow cytometry was used to assess cell cycle events, and scanning electron microscopy (SEM) was used to evaluate the interaction between cells and the biomaterial. The results revealed a statistically significant increase in cell metabolic activity in the laser group compared with the control group at 24 hours (p <0.05) and 48 hours (p <0.001), as indicated by the Alamar blue assay. The Live/Dead assay also revealed a greater density of viable cells in the laser group. The cell cycle analysis revealed a significant increase in the number of cells in the proliferative phase (G2/M) in the laser group compared with the control group (p <0.001). The SEM images demonstrated that the irradiated group had a greater concentration of cells while still maintaining their cell shape and projections. This study demonstrated that photobiomodulation can increase the viability and proliferation of periodontal stem cells cultured on PLA scaffolds, suggesting the potential of this protocol for future studies on periodontal tissue engineering., (© 2024. The Author(s), under exclusive licence to Springer-Verlag London Ltd., part of Springer Nature.)