Barium ferrite nanoparticles (BaFeNPs) were investigated as vehicles for Ra radionuclide in targeted α-therapy. BaFe nanoparticles were labeled using a hydrothermal Ba cations replacement by Ra with yield reaching 61.3 ± 1.8%. Radiolabeled nanoparticles were functionalized with 3-phosphonopropionic acid (CEPA) linker followed by covalent conjugation to trastuzumab (Herceptin). Thermogravimetric analysis and radiometric method with the use of [I]-labeled trastuzumab revealed that on average 19-21 molecules of trastuzumab are attached to the surface of one BaFe-CEPA nanoparticle. The hydrodynamic diameter of BaFe-CEPA-trastuzumab conjugate is 99.9 ± 3.0 nm in water and increases to 218.3 ± 3.7 nm in PBS buffer, and the zeta potential varies from +27.2 ± 0.7 mV in water to -8.8 ± 0.7 in PBS buffer. The [Ra]BaFe-CEPA-trastuzumab radiobioconjugate almost quantitatively retained Ra (>98%) and about 96% of Bi and 94% of Pb over 30 days. The obtained radiobioconjugate exhibited high affinity, cell internalization and cytotoxicity towards the human ovarian adenocarcinoma SKOV-3 cells overexpressing HER2 receptor. Confocal studies indicated that [Ra]BaFe-CEPA-trastuzumab was located in peri-nuclear space. High cytotoxicity of the [Ra]BaFe-CEPA-trastuzumab bioconjugate was confirmed by radiotoxicity studies on SKOV-3 cell monolayers and 3D-spheroids. In addition, the magnetic properties of the radiobioconjugate should allow for its use in guide drug delivery driven by magnetic field gradient.