Magnetite (Fe₃O₄) nanoparticles exhibit complex charge transport behavior governed by mixed-valence electronic structure and defect chemistry. In this study, Fe₃O₄ nanoparticles were synthesized via a controlled co-precipitation method under an inert atmosphere to preserve Fe²⁺ states and suppress secondary phase formation. X-ray diffraction confirms the formation of a nanocrystalline inverse spinel structure with an average crystallite size of ~16 nm. Morphological analysis reveals quasi-spherical, agglomerated nanoparticles. Photoluminescence spectra exhibit distinct emission bands in the visible and near-infrared regions, attributed to defect-mediated recombination involving oxygen vacancies and localized states. Temperature-dependent electrical measurements reveal non-linear semiconducting behavior with an anomalous decrease in resistivity in the 283–293 K range. This behavior is attributed to defect-assisted percolative conduction and thermally activated small polaron hopping rather than any intrinsic phase transition. The study establishes a direct correlation between defect-induced optical states and electrical transport mechanisms. The findings provide important insights into defect-engineered transport phenomena in Fe₃O₄ nanostructures for potential applications in sensing and electronic devices.