Two novel complexes for Zn2+ and VO2+ ions, were prepared from tridentate dibasic chelating Schiff base ligand. 1-((3,5-di-tert-butyl-2-hydroxybenzylidene)amino)naphthalen-2-ol-5‑sodium sulfonate (DSHN), was the ligand used in this study. Alternative spectral tools were applied to elucidate structural composition of new compounds. Also, geometry optimization for all synthesizes was conducted by Gaussian09 program via DFT method, to obtain optimal structures and essential parameters. Moreover, the biochemical behavior for all synthesizes, was explored based on tested reactivity against various microbial strains and cancer cells (HCT-116, MCF-7, and HepG-2). The two complexes exhibited interestingly anti-proliferative potential against human cancer cell lines. The antioxidant behavior of the two complexes was studied by DPPH and SOD assays. Particularly, Zn(II) and VO(II) complexes presented more enhanced antimicrobial and anticancer features compared to the free ligand (DSHN), with superiority for VO(II) complex. The binding nature of two complexes with calf thymus DNA (ctDNA), was examined by various methods as, spectrophotometry, viscosity and gel electrophoresis. Their binding efficiency with ctDNA was proposed to be just intercalation or replacement mode. This in-vitro assay was confirmed by in-silico simulation versus 1cca, 1jnx, 1smp, 2 h80 and 5ajh as the co-crystals for selected pathogen-proteins, which attribute to microbes and cancer cells.