The tetradentate Schiff base ligand (SB), N,N′-bis-(2-mercaptophenylimine)-2,5thiophenedicarboxaldehyde was prepared via condensation of 2,5-thiophenedicarboxaldehyde with 2-aminothiophenol in a 1:2 molar ratio by conventional method. Additionally, its Co(II), Ni(II), Cu(II) and Zn(II) complexes have been synthesized and fully characterized by elemental analysis, FT-IR, 1 H NMR, 13 C NMR, UV-Vis, ESR, ESI-mass, conductivity and magnetic susceptibility measurements. Spectral studies suggested that, the Schiff base coordinate metal ions through the azomethine N-and deprotonated thiol S-atoms. Based on UV-Vis absorption and magnetic susceptibility data, tetrahedral geometry was assigned for both Co(II) and Zn(II) complexes, whereas on the other hand, square planar geometry for both Ni(II) and Cu(II) complexes. The Schiff base and its metal complexes were screened for their in vitro antimicrobial activity by minimum inhibitory concentration (MIC) method. Free radical scavenging activity of the novel compounds was determined by elimination of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals. In addition, the interactions of the free ligand and its complexes with calf thymus DNA (CT-DNA) were explored using absorption, emission and viscosity measurements techniques.