The synthesis and characterization of a novel Schiff base compound, [2-(1H-indol-3-yl)ethyl]-(4-nitrobenzylidene)amine (2IE4NBA), are described and characterized by 1H, 13C NMR, FT-IR, and UV–VIS spectroscopic analysis. The crystal structure was determined by monoclinic space group P21/c, with lattice parameters a = 15.8372(8) Å, b = 11.0720(5) Å, c = 8.5464(4) Å, β = 97.780(2) °, and Z = 4. The intermolecular interactions in the Schiff base 2IE4NBA were theoretically examined by Hirshfeld surface analysis. The optimized structure, HOMO and LUMO energy gap, mulliken population analysis, and molecular Electrostatic Potential (MEP) of the compound were calculated by Density Functional Theory (DFT) calculation with the B3LYP/6-311G basis set method. In addition, we compared calculated and experimental spectral values. Molecular docking studies indicated that the synthesized compound, 2IE4NBA, interacts with the binding pocket of mutated TP53 with a binding affinity of -8.0 kcal/mol. The docking results demonstrated favourable molecular-level interactions with the anticancer drug target, exhibiting strong interactions with active site residues. In vitro studies revealed that the compound exhibited the best minimum inhibitory concentration (MIC) value against Staphylococcus aureus and demonstrated an effect on MDA-MB-231 cancer cell lines. This study investigated its potential as a breast cancer therapeutic agent. 2IE4NBA exhibited promising anti-breast cancer activity against the MDA-MB-231 breast cancer cell line, as confirmed by MTT assay.