The great interest in using nanoparticles (NPs) for biomedical applications is transversal to various materials despite the poorly understood correlation between their physicochemical properties and effects on the immune system. NPs, such as gold and Fe3O4, are generally regarded as safe, but the immunotoxicological profile of Au/Fe3O4 composite NPs with different physicochemical properties is not well documented. This study investigated the biological impact of Au/Fe3O4 composite NPs with different morphologies (spherical core-shell and flower-like) and shell composition in vitro to analyze their potential cytotoxic effects and inflammatory responses on RAW 264.7 cells. Au/Fe3O4 composite NPs with a flower-like structure (FLNPs) induce a pronounced reduction in cell viability compared with Au/Fe3O4 composite NPs with a spherical core-shell structure (CSNPs). The increased production of reactive oxygen species, which damages cellular membranes, might contribute to the cytotoxicity effect of FLNPs. However, CSNPs presented more RAW 264.7 cell adhesion and uptake than FLNPs. Remarkably, a significant TNF-α release was observed with CSNP treated RAW 264.7 cells other than that of FLNPs. Protein corona analysis revealed the adsorption of a distinct amount and profile of proteins on the surface of CSNPs and FLNPs. Given the similar particle size and ζ-potential of CSNPs and FLNPs under the cell culture condition, results indicate that the impact of Au/Fe3O4 composite NPs on the macrophage activity highly depends on their morphology, shell composition and protein corona profile.