Highly thermal stable dithiophenolato Ti(IV)-chitosan nanocomposite (DBT-CS) was formed from dithiophenolato Ti(IV)-complex (DBT) and chitosan (CS) nanoparticles. As DNA-drugs binding is the key reaction step responsible for the anticancer activity, in vitro DNA binding studies to DBT and DBT-CS were investigated and showed a strong interaction to DNA helix via groove binding. Kinetic stability, affinity and association constants of compounds-DNA were studied for the first time using a stopped-flow technique and showed that the formulation of nanostructure DBT-CS does not only improve the rate of reaction through coordination affinity but also change the binding mechanism. Additionally, the antibacterial investigation showed that DBT and DBT-CS exhibited good antibacterial activity against both Gram-positive and Gram-negative bacteria. They also show high cytotoxicity against HepG2 human liver cancer cell. Although DBT-DNA complex is kinetically more stable than that of DBT-CS, the latter showed better antibacterial and cytotoxic activities which in agreement with the binding constants.