The assembly of magnetic cores into regular structures may notably influence the properties displayed by a magnetic colloid. Here, key synthesis parameters driving the self-assembly process capable of organizing colloidal magnetic cores into highly regular and reproducible multi-core nanoparticles are determined. In addition, a self-consistent picture that explains the collective magnetic properties exhibited by these complex assemblies is achieved through structural, colloidal, and magnetic means. For this purpose, different strategies to obtain flower-shaped iron oxide assemblies in the size range 25–100 nm are examined. The routes are based on the partial oxidation of Fe(OH)2, polyol-mediated synthesis or the reduction of iron acetylacetonate. The nanoparticles are functionalized either with dextran, citric acid, or alternatively embedded in polystyrene and their long-term stability is assessed. The core size is measured, calculated, and modeled using both structural and magnetic means, while the Debye model and multi-core extended model are used to study interparticle interactions. This is the first step toward standardized protocols of synthesis and characterization of flower-shaped nanoparticles.