The surface activation of multifunctional nanoparticles (MNPs) with peptide ligands directing their targeting to cancer cells is an emerging research area in nanobiotechnology. In this paper, water-soluble MNPs have been synthesized and functionalized with an scFv antibody variant specific toward the HER2 receptor overexpressed in several breast cancer cell lines. The scFv was genetically engineered to introduce a cysteine residue inside the loop sequence bridging the VH and VL lobes of the molecule and a histidine tag at the C-terminus in the VL fragment. The Cys and 6 × His functionalities were exploited as orthogonal reactive groups driving the scFv conjugation to MNPs. In this way, scFv positioning on the MNP surface was forced into two different orientations depending on the molecular binding site used for conjugation. The resulting scFv-functionalized MNP1 and MNP2, respectively, were assessed as to their labeling efficiency and selectivity to HER2-positive MCF7 cells. We demonstrate that, while both MNP1 and MNP2 were selective for HER2, there is a remarkable preference for scFv presentation with VH and VL lobes concurrently available for receptor recognition (MNP1) in terms of cell binding efficiency, suggesting that ligand orientation may strongly affect cell binding from MNPs.