This study aims to investigate the effect of new natural photosensitizers (PS) (based on oregano essential oil, curcuma extract, and arnica oil) through in vitro cytotoxicity and biological tests in rat-induced periodontal disease, treated with photodynamic therapy (aPDT). The cytotoxicity of PS was performed on human dental pulp mesenchymal stem cells (dMSCs) and human keratinocyte (HaCaT) cell lines. Periodontal disease was induced by ligation of the first mandibular molar of 25 rats, which were divided into 5 groups: control group, periodontitis group, Curcuma and aPDT-treated group, oregano and aPDT-treated group, and aPDT group. The animals were euthanized after 4 weeks of study. Computed tomography imaging has been used to evaluate alveolar bone loss. Hematological and histological evaluation showed a greater magnitude of the inflammatory response and severe destruction of the periodontal ligaments in the untreated group.. For the group with the induced periodontitis and treated with natural photosensitizers, the aPDT improved the results; this therapy could be an important adjuvant treatment. The obtained results of these preliminary studies encourage us to continue the research of periodontitis treated with natural photosensitizers activated by photodynamic therapy.