Green synthesis of nanoparticles is the method with eco-friendly, cost-effectiveness, and ease of resource availability approach. Nowadays, magnetic nanoparticles need increased due to its use in magnetic sensing, medical imaging, waste water treatment, and antibiotic drugs. In this report, the ecofriendly green synthesis of magnetic iron oxide nanoparticles were efficiently synthesized by using Murraya koenigii leaves extract. UV-visible spectrum revealed the surface Plasmon resonance band at 240 nm and analyzed the formation of iron oxide nanoparticles. X-ray diffraction pattern determined its high crystalline nature with strong intense peaks. Fourier Transform Infra-Red spectrum illustrated the Fe and O bonding stretching vibration. The particle size distribution graph showed the formed particles are in nanometer range. Transmission Electron Micrographs realized the spherical shaped iron oxide nanoparticles with its size at 10–25 nm. The energy dispersive X-ray spectrum and mapping analysis revealed the purity of prepared nanoparticles with only Fe and O presence. The vibrating sample magnetometer analysis showed the paramagnetic behavior of prepared magnetic iron oxide nanoparticles. The antimicrobial assay revealed potent inhibition of iron oxide nanoparticles on various human pathogens. The antioxidant, anti-inflammatory and anti-diabetic assays revealed the biomedical behavior of iron oxide nanoparticles.