A series of new ligands 5-(4′-alkylphenylazo)-3-phenylamino-2-thioxothiazolidin-4-one (HL_n) were synthesized from the coupling of 3-phenylamino-2-thioxothiazolidin-4-one with aniline and its p-derivatives. These ligands and their Co(II) polymeric complexes of the [(Co)₂(L_n)₂(HL_n)(CH₃COO)₂(H₂O)₂]_n have been deduced from elemental analyses, IR, ¹H-NMR, X-ray diffraction and mass spectra as well as magnetic and thermal measurements. IR and ¹H NMR studies reveal that the ligands (HL_n) exist in the tautomeric enol/hydrazo form in both states with intramolecular hydrogen bonding. The important infrared (IR) spectral bands imply that HL_n is coordinated to the metal ion in a monobasic tetradentate via NH (hydrazone), oxygen of the carbonyl group (CO), nitrogen of the NH (3-phenylamine) and thion sulfur (CS) group. The complexes are polymeric, non-electrolytes, paramagnetic and octahedral six-coordinated. The molecular and electronic structures of the investigated compounds (HL_n) were also studied using quantum chemical calculations. The salmon sperm DNA (SS-DNA) binding activity of the ligands (HL_n) was studied by absorption spectra. The interaction between ligands (HL_n) and SS-DNA shows hypochromism effect coupled with obvious bathochromism. The values of binding constant are correlated with Hammett_s constant (σ^R). The cytotoxic activity of ligands (HL_n) and their Co(II) complexes were tested against two human cancer HePG-2 (Hepatocellular carcinoma) and MCF-7 (breast cancer). The antioxidant activities of ligands (HL_n) and their Co(II) complexes were performed by ABTS method. The antimicrobial activity of ligands HL_n and their Co(II) complexes were tested against Gram negative bacteria (Escherichia coli), Gram positive bacteria (Staphylococcus aureus) and yeast (Candida albicans).