The control of toxoplasmosis, a rampant one health disease, has been focussed on conventional antitoxoplasmic agents with their adverse outcomes, including serious side effects, treatment failure and emergence of drug resistant strains. Nanobiotechnology may provide a strong impetus for versatile alternative therapies against toxoplasmosis. Bionanofactory Ochrobactrum sp. strain CNE2 was recruited for the biosynthesis of functionalized magnetite iron nanoparticles (MNPs) and nanozerovalent iron (nZVI) under aerobic and anaerobic conditions and their therapeutic efficacy was evaluated against acute toxoplasmosis in murine model. The formation of self-functionalized spherical nanoparticles varied in size, identity and surface properties were substantiated. Mice were orally administered 20 mg/kg of each formulation on the initial day of infection and continued for seven consecutive days post infection (PI). Parasitological, ultrastructural, immunological, and biochemical studies were performed for assessment of therapeutic activity of biogenic iron nanoparticles (INPs). Parasitologically, MNPs showed the highest antitoxoplasmic efficacy in terms of 96.82% and 91.87% reduction in mean tachyzoite count in peritoneal fluid and liver impression smears, respectively. Lesser percentage reductions were recorded in nZVI-treated infected subgroup (75.44% and 69.04%). In addition, scanning electron microscopy (SEM) examination revealed remarkable reduction in size and extensive damage to the surface of MNPs-treated tachyzoites. MNPs-treated infected mice revealed a statistically significant increase in the serum levels of both interferon gamma (IFN-γ) to 346.2 ± 4.6 pg/ml and reduced glutathione (GSH) to 8.83 ± 0.30 mg/dl that subsequently exerted malondialdehyde (MDA) quenching action. MNPs showed a superior promising antitoxoplasmic activity with respect to both spiramycin (SPI) and nZVI. To best of our knowledge, this is the first study of a bio-safe oral iron nanotherapeutic agent fabricated via an eco-friendly approach that offers promising potential against acute experimental toxoplasmosis.