A series of N-substituted isatin thiosemicarbazone ligands (L1–L5) and their nickel(II) complexes [Ni(L)₂] (1–5) were synthesized and characterized by elemental analyses and UV-Visible, FT-IR, ¹H & ¹³C NMR, and mass spectroscopic techniques. The molecular structure of the ligands (L1 and L2) and complex 1 was confirmed by single crystal X-ray crystallography. The single crystal X-ray structure of 1 showed distorted octahedral geometry. The interaction of calf thymus (CT) DNA and bovine serum albumin (BSA) with the nickel(II) complexes was explored using absorption and emission spectral methods. A DNA cleavage study showed that the complexes cleaved DNA without any external agents. The alterations in the secondary structure of the protein by the nickel(II) complexes (1–5) were confirmed by synchronous and three dimensional fluorescence spectroscopic studies. The interaction of the complexes with DNA/protein also has been supported by molecular docking studies. An in vitro cytotoxicity study of the complexes found significant activity against human breast (MCF7) and lung (A549) cancer cell lines, with the best results for complexes 4 and 2 respectively, where the IC50 value is less than 0.1 μM concentration.