The effects of electrostatic interaction between the hen egg white lysozyme (HEWL) and the functionalized iron oxide nanoparticles (IONPs) have been investigated using several techniques, e.g., CD, DSC, ζ-potential, UV–visible spectroscopy, DLS, TEM. Nanoparticles (IONPs) were functionalized with three hydrophilic ligands, viz., poly(ethylene glycol) (PEG), trisodium citrate (TSC) and sodium triphosphate (STP); where both TSC and STP contain Na⁺ counter ions. It has been observed that the secondary structure of HEWL was not affected by PEG functionalized IONPs, but was partially and almost completely perturbed by TSC and STP functionalized IONPs, respectively. The perturbation of the secondary structure was irreversible. We have predicted an interaction model to explain the origin of perturbation of HEWL structure. We have also investigated the stability of nanoparticles dispersions after interaction with HEWL and used the DLVO theory to explain results.