Magnetic iron oxide nanoparticles (IONPs) have been widely used for various biomedical applications such as magnetic resonance imaging and drug delivery. However, their potential toxic effects, including genotoxicity, need to be thoroughly understood. In the present study, the genotoxicity of IONPs with different particle sizes (10, 30 nm) and surface coatings (PEG, PEI) were assessed using three standard genotoxicity assays, the Salmonella typhimurium reverse mutation assay (Ames test), the in vitro mammalian chromosome aberration test, and the in vivo micronucleus assay. In the Ames test, SMG-10 (PEG coating, 10 nm) showed a positive mutagenic response in all the five test bacterial strains with and without metabolic activation, whereas SEI-10 (PEI coating, 10 nm) showed no mutagenesis in all tester strains regardless of metabolic activation. SMG-30 (PEG coating, 30 nm) was not mutagenic in the absence of metabolic activation, and became mutagenic in the presence of metabolic activation. In the chromosomal aberration test, no increase in the incidence of chromosomal aberrations was observed for all three IONPs. In the in vivo micronucleus test, there was no evidence of increased micronuclei frequencies for all three IONPs, indicating that they were not clastogenic in vivo. Taken together, our results demonstrated that IONPs with PEG coating exhibited mutagenic activity without chromosomal and clastogenic abnormalities, and smaller IONPs (SMG-10) had stronger mutagenic potential than larger ones (SMG-30); whereas, IONPs with SEI coating (SEI-10) were not genotoxic in all three standard genotoxicity assays. This suggests that the mutagenicity of IONPs depends on their particle size and surface coating.