Background <p>Autogenous tooth bone graft is being evaluated as an alternative to existing bone grafts for supporting bone healing and may enable new regenerative therapies. This study investigated its effects on the proliferation, migration, attachment, and mineralized tissue formation of human osteosarcoma cells (Saos-2), gingival fibroblasts (hGF), peripheral blood monocytes (THP-1), and periodontal ligament fibroblasts (hPDLF) via an in vitro study.</p> Methods <p>The cell viability in autogenous tooth bone graft, allograft, xenograft, and synthetic bone graft groups was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). Calcification was assessed via Alizarin Red staining and osteocalcin activity test. The cell adhesion and morphology were examined using scanning electron microscopy (SEM). The cell proliferation and migration outcomes at 24, 48, and 72&#xa0;h were evaluated using analysis of variance, and Tukey’s test was used for post-hoc comparisons.</p> Results <p>The highest proliferation rates of the Saos-2, hGF, THP-1, and hPDLF cells were observed in the autogenous tooth bone graft group at 72&#xa0;h. No significant differences in cell migration were observed between the graft groups. Furthermore, calcification in the Saos-2 cells was higher in the autogenous tooth bone graft group than in other graft groups. SEM revealed that the cell adherence, spread, and morphology were similar in the autogenous tooth bone graft, allograft, xenograft, and synthetic bone graft groups.</p> Conclusions <p>Our study results demonstrate that autogenous tooth bone graft positively affects cell viability and mineralized tissue formation.</p> Graphical Abstract <p></p>