Metallic nanoparticles have received a significant amount of reflection over a period of time, attributed to their electronic, specific surface area, and surface atom properties. The biogenic synthesis of iron oxide nanoparticles (FeONPs) is demonstrated in this study. The green synthesis of metallic nanoparticles (NPs) is acquiring considerable attention due to its environmental and economic superiorities over other methods. sp. L-2 extract was employed as a reducing agent, and iron chloride hexahydrate (FeCl·6HO) was used as a substrate for the biogenic synthesis of FeONPs. Different spectral methods were used for the characterization of the biosynthesized FeONPs, ultraviolet-visible (UV-Vis) spectroscopy gave a surface plasmon resonance (SPR) peak of FeONPs at 300 nm; Fourier transform infrared (FTIR) spectral analysis was conducted to identify the functional groups responsible for both the stability and synthesis of FeONPs. The morphology of the FeONPs was investigated using scanning electron microscopy (SEM), which shows a nearly spherical shape, and an X-ray diffraction (XRD) study demonstrated their crystalline nature with a calculated crystallinity size of 23 nm. The zeta potential (ZP) and dynamic light scattering (DLS) measurements of FeONPs revealed values of -8.50 mV, suggesting appropriate physical stability. Comprehensive in-vitro pharmacogenetic properties revealed that FeONPs have significant therapeutic potential. FeONPs have been reported to have potential antibacterial and antifungal properties. Dose-dependent cytotoxic activity was shown against promastigotes (IC50: 10.73 µg/mL) and amastigotes (IC50: 16.98 µg/mL) using various concentrations of FeONPs. The cytotoxic potential was also investigated using brine shrimps, and their IC50 value was determined to be 34.19 µg/mL. FeONPs showed significant antioxidant results (DPPH: 54.7%, TRP: 49.2%, TAC: 44.5%), protein kinase (IC50: 96.23 µg/mL), and alpha amylase (IC50: 3745 µg/mL). The biosafety of FeONPs was validated by biocompatibility tests using macrophages (IC50: 918.1 µg/mL) and red blood cells (IC50: 2921 µg/mL). In conclusion, biogenic FeONPs have shown potential biomedical properties and should be the focus of more studies to increase their nano-pharmacological significance for biological applications.