Periodontitis is an oral chronic inflammatory disease induced by microorganisms that can destroy tooth‑supporting structures. Human β‑defensin‑3 (HBD‑3) is a type of endogenous antimicrobial peptide that inhibits a broad spectrum of microorganisms. The objectives of the present study were to transfect human periodontal ligament cells (HPDLCs) and human bone marrow stromal cells (HBMSCs) with lentivirus containing the HBD‑3 gene, determine the transfection efficiency, and investigate the antimicrobial activity of the experimental cells against periodontal pathogens. Fluorescence microscopy was used to calculated the transfection efficiency. Western blot analysis and ELISA were conducted to confirm the expression of HBD‑3 at the protein level. The effect of the HBD‑3 gene on the antimicrobial activity of the cells were demonstrated by antimicrobial tests. The results of the present study demonstrated that the transfected HPDLCs and HBMSCs stably expressed HBD‑3. In addition, periodontal pathogens and caries‑causing bacteria were susceptible to the antimicrobial activity of the cells. Both HPDLCs and HBMSCs hold potential for use as seeding cells in cell‑ and gene‑based therapies for periodontal disease. The lentiviral vector containing HBD‑3 resulted in broad‑spectrum antimicrobial activity against a variety of oral organisms, and could potentially be applied in the treatment of oral infectious diseases, including periodontitis.