Due to their high chemical affinity towards metal ions, chelating agents (CAs) have been used for decades for water purification, but also for protection against metal intoxication and in nanomedicine as linking molecules at the surface of nanoparticles. However, this strong chemical activity could also impact their colloidal behavior, which is essential for biomedical applications. Therefore, we coated iron oxide nanoparticles (IONPs) with four CAs, differing in their number of active chemical groups, with variations from 2 to 5 dents containing carboxylic groups: iminodiacetic acid (IDA), nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA). We found large differences between the CA-coated IONPs depending on the CA nature, especially regarding their agglomeration state and colloidal behavior, but also consequently their cellular uptake. Surprisingly, although CAs have been widely used for biomedical applications, CA-coated IONPs, especially IDA-and EDTA-coated IONPs, showed non-negligible toxicity. Moreover, for their application as contrast agents for MRI, we found that CA-coated IONPs displayed high r(2) relaxivities, which differed according to their agglomeration state. Overall, our study suggests that CAs, depending on their chemical nature, can induce agglomeration and toxicity, which could be harmful in a clinical setting.