The new NO bi-dentate imine ligand namely (2-[(3-Nitro-benzylidene)-amino]-benzoic acid) (NBA) imine ligand (L) and their Pd(II) and VO(II) complexes have synthesized and characterized utilizing different physiochemical methods including elemental analyses, FT-IR, NMR, molar conductance and magnetic susceptibility measurements. The stability constant of these complexes has also determined. The studies of these complexes were found to have [M (L)] composition where the ligand acts as a bi-dentate and coordinates with the various metal ions through azomethine and one oxygen atom. Moreover, the suggested geometrical structure of Pd(II) and VO(II) complexes are square planar and a distorted square pyramidal, respectively. DFT calculations for the different metal-complexes were studied and show a good agreement with the experiment data. The minimum inhibitory concentration method has been applied to evaluate the in vitro antimicrobial activity. Furthermore, the interaction of calf thymus DNA with the metal imine chelates has assessed and Pd(II) complex showed the strongest interaction with DNA for a binding constant value of (9.72 x 104 kcal mol-1). The cytotoxic activity of the new metal chelates has evaluated against 3 human cancer cell lines (hepatic carcinoma HepG2 cells, breast carcinoma cells MCF-7, and colon carcinoma cells HCT-116). It has found that NBAPd complex exhibited a significant decrease in the time and dose of the cell viability compared to the reference drug vinblastine. Antioxidant activity of the tested compounds was determined and compared to vitamin C as a standard drug. It has found that the metal complexes exhibit higher activity than that of the free ligand. The finding from this investigation revealed that the new compounds are considered to be prospective antibiotic and anticancer agents.