Abstract: Manganese-substituted magnetite ferrofluids (FFs) Mn_xFe_1−xFe₂O₄ (x = 0–0.8) were prepared in this work through a chemical co-precipitation reaction. The controlled growth of FF nanomaterials for antibacterial activities is challenging, and therefore, very few reports are available on the topic. This research focuses on stabilizing aqueous FFs with the tetramethylammonium hydroxide surfactant to achieve high homogeneity. Morphological characterization reveals nanoparticles of 5–11 nm formed by the chemical reaction and nanocrystalline nature, as evident from structural investigations. Mn-substituted magnetic FFs are analyzed for their structural, functional, and antibacterial performance according to the Mn-substituent content. Optical studies show a high blue shift for Mn²⁺-substituted Mn_xFe_1−xFe₂O₄ with the theoretical correlation of optical band gaps with the Mn content. The superparamagnetic nature of substituted FFs causes zero coercivity and remanence, which consequently influence the particle size, cation distribution, and spin canting. The structural and functional performance of the FFs is correlated with the antibacterial activity, finally demonstrating the highest inhibition zone formation for Mn_xFe_1−xFe₂O₄ FFs.