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Polymorphic Form-I and Form-II of (E)-4-Bromo-2-[(phenylimino)methyl]phenol [4B2PMP] commonly known as N-salicylidene have been identified. Form-I and Form-II are synthesized in the temperature at 283 K and 333 K respectively. In both forms, the molecule shows some resembling structures in the geometrical arrangements but differing in properties. Structural affirmation accomplished by well-defined strong Bragg's diffraction pattern through powder X-Ray diffraction patterns with assigning Nuclear magnetic resonance chemical shifts and its data analysis. Intramolecular OH⋯N weak hydrogen bond between the -OH phenol group and the imine =N- group found in both forms but differing in torsion angles. Optical behavior studied through Diffuse-Reflectance UV–VIS spectroscopy. The Molecular docking approach exhibits ligand (4B2PMP) – protein (DNA Gyrase B) interaction against Escherichia coli shows variations between the two forms which is due to the unique property of crystal polymorphism. Polymorphism makes 4B2PMP-Form II crystal have the highest binding affinity than Form I crystal.