(E)-4-Chloro-2-[(pyridin-2-ylimino)methyl]phenol was synthesized in the reaction of 2-aminopyridine with 5-chlorosalicylaldehyde. The structure of compound was investigated by FTIR, UV-Vis, 1H-NMR, C-13-NMR and X-ray data. In addition, characterization of the compound was realized using theoretical quantum mechanical calculations and experimental spectroscopic methods. The molecular structure of the compound was confirmed using X-ray single-crystal data, NMR, FTIR and UV-Vis, which were in good agreement with the structure predicted by the theoretical calculations using the density functional theory (DFT). Moreover, the antimicrobial activity of the compound was investigated against some bacteria and yeast cultures by the broth microdilution test. UV-Vis spectroscopy studies of the interactions between the Schiff base and calf thymus DNA (CT-DNA) showed that the compound interacts with CT-DNA via electrostatic binding. The colorimetric response of the compound receptors was investigated before and after the addition of an equivalent amount of each anion to evaluate anion recognition properties.