Since cancer is a continuously increasing concern for the general population, more efficient treatment alternatives ought to be developed. In this regard, a promising direction is represented by the use of magnetite nanoparticles (MNPs) to act both as a nanocarrier for the targeted release of antitumoral drugs and as hyperthermia agents. Thus, the present study focused on improving the control upon the outcome properties of MNPs by using two synthesis methods, namely the co-precipitation and microwave-assisted hydrothermal method, for the incorporation of usnic acid (UA), a natural lichen-derived metabolite with proven anticancer activity. The obtained UA-loaded MNPs were thoroughly characterized regarding their morpho-structural and physicochemical properties through X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FT-IR), dynamic light scattering (DLS) and zeta potential, scanning electron microscopy (SEM), and vibrating sample magnetometry (VSM). Results demonstrated the formation of magnetite as the unique mineralogical phase through both types of synthesis, with increased uniformity regarding the drug loading efficiency, size, stability, and magnetic properties obtained through the microwave-assisted hydrothermal method. Furthermore, the cytotoxicity of the nanostructures against the HEK 293T cell line was investigated through the XTT assay, which further proved their potential for anticancer treatment applications.