Oxovanadium complexes (5a–5g) with fluoroquinolone ligands and azodye rhodanine (4-hydroxy-5-((4-hydroxyphenyl)diazenyl)thiazole-2(3H)-thione) were synthesized and the effect of different groups was studied, which displayed various biological and medicinal activities. The newly formed compounds were characterized using ESI-MS, UV-vis, IR, ESR spectroscopy, elemental analysis and molar electric conductivity. The in vitro antibacterial activity of the complexes against three Gram negative and two Gram positive microorganisms was studied and compared to the activity of free ligand and it shows considerable results. The minimum inhibitory concentration (MIC) of the compounds against Mycobacterium tuberculosis was determined and among all the complexes, 5c and 5d showed potency. The in vitro cytotoxicity of the complexes was determined with a brine shrimp bioassay and the LD₅₀ obtained is in the range of 4–12 μg mL⁻¹. The DNA binding activity was studied using UV-vis titration and viscosity measurements, and 5c has the highest binding constant value (6.91 × 10⁵ M⁻¹). DNA nuclease activity was studied using the agarose gel electrophoresis method and the compounds show 70–80% cleavage.