Magnetic nanoparticles occupy a significant class of inorganic nanoparticles with its very promising application in biosensors and bio-imaging. Physical and chemical methods have been used conventionally to prepare magnetic nanoparticles. However, these methods have its own limitations, which redirected recent research towards safe, clean and eco-friendly green synthesis approach. In our study, green magnetic iron oxide nanoparticles (FeNP DG) were prepared by using aqueous Desmodium gangeticum root extract as a reducing as well as a capping agent. Nanoparticles used in different biological milieu has to be explored for their toxicity before its application. Thus prepared nanoparticles were evaluated for its nephrotoxicity in Wistar rats (100 mg/kg b.wt), in renal epithelial cells, LLC PK1 (1 mg/ml) and isolated mitochondria (0.25, 0.5, 1 mg/ml) and compared it with the conventionally prepared iron oxide nanoparticles (FeNP Chem). Our study demonstrate that chemically prepared FeNPs are toxic to kidney and its epithelial cells.On the other hand, when the same nanoparticles were prepared by green route (FeNP DG) exhibited minimum toxicity measured via the renal markers in blood and urine along with cytotoxicity assay in LLC PK1 cells. But at mitochondrial level, both FeNP Chem and FeNP DG were found to be toxic to the organelle.