Two novel VO(II) and Mn(II) imine chelates derived from the condensation of o-phenylenediamine (PN) with 3-ethoxysalicylaldehyde(ES) were synthesized. The prepared ESPN imine ligand and its chelates were investigated via different analytical and physicochemical tools. Correlation between all the obtained results, the parent ligand behaves as tetra-dentate ONNO ligand and coordinates to Mn(II) and VO(II), in octahedral and square pyramidal geometry, respectively. Also, the prepared compounds were screened for their antimicrobial activities on different pathogenic bacteria and fungi. The results show that the complexes have a stronger antimicrobial activity in comparison with its ligand. Moreover, the interaction of the prepared metal chelates with CT-DNA was detected utilizing spectral studies, viscosity and gel electrophoreses measurements. The obtained results clearly demonstrate that, the binding affinity with CT-DNA for ESPN > ESPNV complex. Furthermore, the cytotoxicactivity of the tested compounds on human colon carcinoma cells, hepatic cellular carcinoma cells and breast carcinoma cells has been examined. From these results it was found that the investigated complexes have effective cytotoxicity against growth of carcinoma cells with respect of its imine ligand.