Herein, we report the synthesis of (E)-2-(4-methoxybenzylidene) aminophenol (3a) and its mono/dihalo substituted aryl analogues (3b, 3c, and 3d), achieved by the condensation of 2-aminophenol derivatives with 4-methoxybenzaldehyde. The structures of these Schiff bases were confirmed using a suite of spectroscopic and analytical techniques. Single-crystal X-ray diffraction was used to determine the solid-state molecular conformation and intermolecular interactions that exist in the crystal packing of the resulting Schiff bases. Interestingly, the presence of a meta-chloro substituent in 3b increases the dihedral angle when compared to 3a, suggesting that the molecular conformation is influenced by the acid dissociation constants (pKa) of the aniline precursors. Cytotoxicity assays against HeLa cancer cells and HEK293 healthy cells demonstrated dose-dependent activity, revealing a correlation between electronic effect of the halo substituents, molecular conformation and biological activity. The dihalogenated Schiff base 3d showed the highest antiproliferative activity against the Hela tumour cell (IC50 = 24.96 ± 0.15 μM) while the non-halogenated Schiff base 3a showed the highest selectivity index (SI) towards the healthy HEK293 cells (1.926). Based on the energies of the various intermolecular interactions in the crystal packing of the reported Schiff bases, the ratio of the electrostatic to dispersion energy contribution (Eele/Edis) for the intermolecular π-related interactions appear to significantly influence the molecular conformation and biological activity of the compounds. Higher Eele/Edis ratios promote planar conformations and greater SI, while lower ratios lead to more twisted conformations and increased cytotoxic potency. The findings from both the electronic effect and the structural conformation of the Schiff base compounds comulatively suggest a unique approach to drug design by optimizing the balance between the electronics of the halo substituents, electrostatic and dispersive energy contributions in specific intermolecular interactions. These can fine tune both the SI and cytotoxicity towards cancerous HeLa cells.