We synthesized manganese ferrite (MnFe₂O₄) nanoparticles of different sizes by varying pH during chemical co-precipitation procedure and modified their surfaces with polysaccharide chitosan (CS) to investigate characteristics of hyperthermia and magnetic resonance imaging (MRI). Structural features were analyzed by X-ray diffraction (XRD), high-resolution transmission electron microscopy (TEM), selected area diffraction (SAED) patterns, and Mössbauer spectroscopy to confirm the formation of superparamagnetic MnFe₂O₄ nanoparticles with a size range of 5–15 nm for pH of 9–12. The hydrodynamic sizes of nanoparticles were less than 250 nm with a polydispersity index of 0.3, whereas the zeta potentials were higher than 30 mV to ensure electrostatic repulsion for stable colloidal suspension. MRI properties at 7T demonstrated that transverse relaxation (T₂) doubled as the size of CS-coated MnFe₂O₄ nanoparticles tripled in vitro. However, longitudinal relaxation (T₁) was strongest for the smallest CS-coated MnFe₂O₄ nanoparticles, as revealed by in vivo positive contrast MRI angiography. Cytotoxicity assay on HeLa cells showed CS-coated MnFe₂O₄ nanoparticles is viable regardless of ambient pH, whereas hyperthermia studies revealed that both the maximum temperature and specific loss power obtained by alternating magnetic field exposure depended on nanoparticle size and concentration. Overall, these results reveal the exciting potential of CS-coated MnFe₂O₄ nanoparticles in MRI and hyperthermia studies for biomedical research.