Magnetic iron oxide nanoparticles (MIONPs) were placed in the spotlight lately due to their excellent biocompatibility and the possibility to tailor their magnetic properties making them useful in a plethora of bioapplications, including magnetic resonance imaging and targeted nanoplatforms for anticancer drugs delivery. The aim of the present study consisted in achieving a toxicological profile of the biocompatible colloidal suspension of iron oxide nanoparticles coated with a double layer of oleic acid (Fe₃O₄ @OA) obtained by combustion method by performing in vitro (on human keratinocytes-HaCaT and human and murine melanoma cells) and in ovo studies on chick chorioallantoic membrane (HET-CAM assay). The colloidal suspension obtained proved to be stable in phosphate buffer saline and the size of the nanoparticles were in the range of 30 nm, an optimum size for biomedical applications. Fe₃O₄ @OA colloidal suspension reduced viability of human keratinocytes only at concentrations higher than 25 μg/mL, whereas in the case of melanoma cells the effect was observed at lower doses (starting with 10 μg/mL). An interesting phenomena was detected at the highest concentration tested (50 μg/mL) to all cell lines, more precisely, a particular enucleation process associated only with Fe₃O₄ @OA colloidal suspension stimulation. The irritant potential data evaluated by HET-CAM assay indicated the following hierarchy: Fe₃O₄ < Fe₃O₄ @OA < OA. Our results provide relevant information regarding the mechanism of action of Fe₃O₄ @OA that needs elucidation by future in vitro and in vivo studies.