Chemical oxidative polymerization was employed to synthesize pure poly (ortho-toluidine) (POT). The synthesized POT was characterized using XRD, FT-IR, and SEM analyses. Additionally, the samples were subjected to biological evaluation. The antibacterial activity of POT was tested against both Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria. Notably, E. coli displayed significant susceptibility to POT's antibacterial effects, especially at higher concentrations. Furthermore, in vitro anticancer experiments were conducted on MG-63 bone cancer cells to assess the cytotoxicity of POT. Pristine POT exhibited the highest level of cytotoxicity, as evidenced by an IC50 value of 83.15 g/ml. In summary, this investigation highlights the robust antibacterial and anticancer activities of POT. These findings suggest potential applications of POT in areas such as tissue engineering scaffolds, anticancer therapies, and various biomedical uses.