The use of non-toxic synthesis of iron oxide nanoparticles (FeO NPs) by an aqueous plant extract has proven to be a viable and environmentally friendly method. Therefore, the present investigation is based on the FeO NPs synthesis by means of FeCl3·6H2O as a precursor, and the plant extract of Nephrolepis exaltata (N. exaltata) serves as a capping and reducing agent. Various techniques were used to examine the synthesized FeO NPs, such as UV-Visible Spectroscopy (UV-Vis), Fourier Transform Infrared Spectroscopy (FT-IR), X-ray Diffraction (XRD), Scanning Electron Microscopy (SEM), and Energy Dispersive X-ray (EDX). The FT-IR studies were used to identify different photoactive biomolecules at 3285, 2928, 1415, 1170, and 600 cm−1 in the wavenumber range from 4000 to 400 cm−1, indicating the -OH, C-H, C-O, C-C, and M-O groups, respectively. The XRD examination exhibited crystallinity, and the average diameter of the particle was 16 nm. The spherical nature of synthesized FeO NPs was recognized by SEM images, while the elemental composition of nanoparticles was identified by an EDX spectrophotometer. The antiplasmodial activity of synthesized FeO NPs was investigated against Plasmodium parasites. The antiplasmodial property of FeO NPs was evaluated by means of parasite inhibitory concentration, which showed higher efficiency (62 ± 1.3 at 25 μg/mL) against Plasmodium parasite if compared to plant extracts and precursor. The cytotoxicity of FeO NPs was also assessed in human peripheral blood mononuclear cells (PBMCs) under in vitro conditions. The lack of toxic effects through FeO NPs keeps them more effective for use in pharmaceutical and medical applications.