Three aroylhydrazone ligands ((Z)-N'-([2,2'-bithiophen]-5-ylmethylene)-2-hydroxybenzohydrazide, HL1; (Z)-N'-([2,2'-bithiophen]-5-ylmethylene)-3-hydroxybenzohydrazide, HL2; and (Z)-N'-([2,2'-bithiophen]-5-ylmethylene)-4-hydroxybenzohydrazide, HL3) and their complexes with nickel (Ni(L1)(2), 1; Ni(L2)(2), 2; Ni(L3)(2)∙DMF, 3) were synthesized and characterized by ESI-MS, NMR, IR, UV-vis and elemental analysis techniques. The molecular structure of ligand (HL2) and complexes 1-3 was confirmed by single crystal X-ray crystallography. The single crystal X-ray structure of complexes 1-3 showed a distorted square planar geometry around the metal center, and the ligands adopt a bidentate chelating mode. The interaction of calf thymus (ctDNA) with nickel(II) complexes was explored using absorption, emission spectrum, viscosity, and circular dichroism methods. These complexes exhibited moderate affinity for ctDNA through groove binding modes. The most efficient DNA binder was complex 2. The interaction of the complexes with DNA has also been supported by molecular docking study and molecular dynamics simulation. An in vitro cytotoxicity study of the complexes found low activity against human cervical (Hela) and breast (MCF-7) cancer cell lines, with the best results for complex 2, where IC50 values are 86 μM and 92 μM respectively.