First Ni(II) ternary complex using the quinolone antibacterial agent enoxacin (HEn) as ligand and 1,10-phenanthroline as co-ligand has been synthesized and characterized. It is a mononuclear structure, in which enoxacin acts as a bidentate ligand bound to the metal through the ketone oxygen and a carboxylate oxygen atom. The complex exhibited good binding propensity to human and bovine serum albumin proteins having relatively high binding constants (6.40×104 and 7.12×104, respectively). The investigation of the interaction of the complex with calf-thymus (CT) DNA has been performed with UV and circular dichroism (CD) spectroscopies, indicating that they bind to CT DNA probably by the intercalative binding mode. The binding constant ( Kb) of the complex with CT DNA calculated with UV is 2.03×105, which is higher than that of free enoxacin drug (2.09×104) and even higher than that of typical intercalation indicator (1.23×105) of ethidium bromide (EB). Fluorescence competitive studies with EB have revealed that the complex exhibited the ability to displace the DNA-bound EB using the intercalative binding site. In addition, the antimicrobial activity showed that the complex exhibited a little bit good inhibition (MIC=1.843 (g·mL−1) against B. subtilis than free HEn. [ABSTRACT FROM AUTHOR]