Ferrofluid-based manganese (Mn(2+)) substituted superparamagnetic iron oxide nanoparticles stabilized by surface coating with trisodium citrate (MnIOTCs) were synthesized for enhanced hyperthermic activity and use as negative magnetic resonance imaging (MRI) contrast media intended for applications in theranostics. The synthesized MnIOTC materials were characterized based on their physicochemical and biological features. The crystal size and the particle size at the nano level were studied using XRD and TEM. The presence of citrate molecules on the crystal surface of the iron oxide was established by FTIR, TGA, DLS and zeta potential measurements. The superparamagnetic property of MnIOTCs was measured using a vibrating sample magnetometer. Superparamagnetic iron oxide substituted with Mn(2+) with a 3:1 molar concentration of Mn(2+) to Fe(2+) and surface modified with trisodium citrate (MnIO75TC) that exhibited a high T2 relaxivity of 184.6mM(-1)s(-1) and showed excellent signal intensity variation in vitro. Hyperthermia via application of an alternating magnetic field to MnIO75TC in a HeLa cell population induced apoptosis, which was further confirmed by FACS and cLSM observations. The morphological features of the cells were highly disrupted after the hyperthermia experiment, as evidenced from E-SEM images. Biocompatibility evaluation was performed using an alamar blue assay and hemolysis studies, and the results indicated good cytocompatibility and hemocompatibility for the synthesized particles. In the current study, the potential of MnIO75TC as a negative MRI contrast agent and a hyperthermia agent was demonstrated to confirm its utility in the burgeoning field of theranostics.