Periodontal ligament (PDL) has become an elective source of mesenchymal stem cells (PDLSCs) in dentistry. This research aimed to compare healthy PDLSCs (hPDLSCs) and periodontitis PDLSCs (pPDLSCs) to ascertain any possible functional differences owing to their milieux of origin. Cells were tested in terms of colony-forming unit efficiency; multi differentiating capacity; immunophenotype, stemness, and senescent state were studied by flow cytometry, immunofluorescence, and β-galactosidase staining; gene expression using RT-PCR. Both hPDLSCs and pPDLSCs were comparable in terms of their immunophenotype and multilineage differentiation capabilities, but pPDLSCs showed a senescent phenotype more frequently. Thus, a selective small molecule inhibitor of DNA methyltransferase (DNMT), RG108, known for its effect on senescence, was used to possibly reverse this phenotype. RG108 did not affect the proliferation and apoptosis of PDLSCs, and it showed little effect on hPDLSCs, while a significant reduction of both p16 and p21 was detected along with an increase of SOX2 and OCT4 in pPDLSCs after treatment at 100 μM RG108. Moreover, the subset of PDLSCs co-expressing OCT4 and p21 decreased, and adipogenic potential increased in pPDLSCs after treatment. pPDLSCs displayed a senescent phenotype that could be reversed, opening new perspectives for the treatment of periodontitis.