In this study, chitosan–titanium dioxide nanoparticles (CS–TiO₂NPs) were synthesized using an extract of Phyllanthus niruri (L.) and characterized using various spectroscopic and analytical methods. UV–Vis spectroscopy validated the synthesis of nanoparticles, showing a prominent absorption peak at 320 nm, while FT-IR analysis identified the functional groups involved in their reduction and stabilization. XRD patterns showed a crystalline structure with distinct, sharp peaks. HR-TEM analysis indicated that the particles were predominantly spherical, averaging 23 nm in size, while EDX confirmed the presence of titanium and oxygen elements. Biological evaluations indicated that CS–TiO₂NPs exhibit strong multifunctional characteristics. Antioxidant evaluations demonstrated significant activity against DPPH (82.46 ± 1.22 %) and H₂O₂ (79.63 ± 1.24 %) at a concentration of 100 μg/mL. Antibacterial activity was observed against Escherichia coli (18.34 ± 1.1 mm) and Staphylococcus aureus (17.26 ± 1.0 mm). Marked anti-inflammatory effects were recorded against COX-1 (68.53 ± 0.92 %) and COX-2 (78.29 ± 0.96 %). Additionally, CS–TiO₂NPs demonstrated antidiabetic properties by inhibiting α-amylase (68.53 ± 0.92 %) and α-glucosidase (68.53 ± 0.92 %), along with anticholinesterase activity against AChE (71.29 ± 1.83 %) and BChE (78.26 ± 1.74 %). Notably, cytotoxicity tests against HeLa cancer cells showed a reduction in viability to 29.16 ± 0.85 % at 100 μg/mL, confirming their anticancer potential. The molecular docking study demonstrated that ursolic and oleanolic acids showed significant binding affinities, with the 1U54 and 1HCK complexes exhibiting the highest stability. In conclusion, CS–TiO₂NPs synthesized from P. niruri extract demonstrate significant antioxidant, antibacterial, anti-inflammatory, antidiabetic, anticholinesterase, and anticancer activities, indicating their potential biomedical applications. © 2025 Elsevier B.V.