The ever-increasing resistance of plant microbes towards fungicides and bactericides has been causing serious threat to plant production in recent years. For the development of an effective antifungal agent, we introduce a novel hydrothermal protocol for synthesis of chitosan iron oxide nanoparticles (CH-Fe 2 O 3 NPs) using acetate buffer of low pH 5.0 for intermolecular interaction of Fe 2 O 3 NPs and CH. The composite structure and elemental elucidation were carried out by using X-ray power diffraction (XRD), Scanning Electron Microscopy (SEM), Energy Dispersive X-ray (EDX), Transmission Electron Microscopy (TEM), Fourier Transformed Infrared Spectroscopy (FTIR) and Ultraviolet Visible Absorption Spectroscopy (UV-vis spectroscopy). Additionally, antifungal activity was evaluated both In vitro and In vivo against Rhizopus oryzae which is causing fruit rot disease of strawberry. We compared different concentrations (0.25%, 0.50%, 075% and 1%) of CH-Fe 2 O 3 NPs and 50% synthetic fungicide (Matalyxal Mancozab) to figure out suitable concentration for application in the field. XRD analysis showed a high crystalline nature of the NPs with average size of 52 nanometer (nm). SEM images revealed spherical shape with size range of 50-70 nm, whereas, TEM also revealed spherical shape, size ranging from 0 nm to 80 nm. EDX and FTIR results revealed presence of CH on surface of Fe 2 O 3 NPs. The band gap measurement showed peak 317-318 nm for bare Fe 2 O 3 NPs and CH-Fe 2 O 3 NPs respectively. Antifungal activity in both In vitro and In vivo significantly increased with increase in concentration. The overall results revealed high synergetic antifungal potential of organometallic CH-Fe 2 O 3 NPs against Rhizopus oryzae and suggest the use of CH-Fe 2 O 3 NPs against other Phyto-pathological diseases due to biodegradable nature.