Magnetite (Fe₃O₄) nanoparticles (NPs) have widely used in various fields, including in medicine, due to their (super)paramagnetic properties. This requires a thorough evaluation of their possible hazardous effects. However, there is no standard procedure for the preparation of oxidation-prone NPs (such as magnetite) before subjecting them to biological assays. In this study we used Fe₃O₄ NPs (bare and silica-coated) as test samples to compare different preparation methods (ultrasound, centrifugation and filteration of NPs suspensions) based on X-ray and dynamic light scattering analysis and evaluation of microstructure and surface charge. After oxidation and functionalization, all samples retained their superparamagnetic behaviour. The toxicity of NP suspensions obtained by the methods described for Paramecium caudatum ciliates and Sinapis alba plants was evaluated.