Three novel Cr(III),VO(II) and Ni(II) imine complexes derived from the condensation of 2‐aminophenol (AP) with 2‐hydroxynaphthaldehyde (HN) were synthesized. The prepared HNAP imine ligand and its complexes were investigated via various physicochemical tools. The results suggest that the parent ligand behaves as a dibasic tridentate ONO ligand, when coordinated to Cr(III) in octahedral and to Ni(II) in tetrahedral geometry. In the case of VO(II), it coordinates in distorted square pyramidal geometry. Also, the prepared compounds were screened for their antimicrobial activities against pathogenic bacteria, Escherichia coli (−ve), Bacillus subtilis (+ve) and Staphylococcus aureus (+ve), and some types of fungi, Aspergillus niger, Candida glabrata and Trichophyton rubrum. The results indicate that the complexes show a stronger antimicrobial efficiency compared to the pro‐ligand. The interaction of the prepared complexes with calf thymus DNA was investigated using spectral, viscosity and gel electrophoresis measurements. The obtained results clearly demonstrate that the binding affinity with calf thymus DNA follows the order HNAPCr > HNAPV > HNAPNi. The cytotoxic activity of the prepared compounds on human colon carcinoma cells (HCT‐116 cell line), hepatic cellular carcinoma cells (HepG‐2cell line) and breast carcinoma cells (MCF‐7cell line) was examined. From these results it is found that the investigated complexes have potent cytotoxicity against growth of carcinoma cells compared to the corresponding imine pro‐ligand.