Organosolv lignin nanoparticles have been recently evaluated for their use in mineral froth flotation as a flotation reagent, and as a result, the recovery of the target minerals was improved and the selectivity of the process was increased. However, the mechanism of lignin activity in mineral froth flotation is not known. Therefore, this study is the first step in understanding the interaction of organosolv lignin with the mineral surface. As such, the organosolv lignin was characterized by GPC and 31P NMR, where the structural differences between the birch and spruce lignins were determined. The molecular size and lignol unit composition were evaluated. Subsequently, the morphology and size of the organosolv lignin particles were examined for all 4 produced types: BN, BM, SN, and SM. The ζ potential was measured in the pH range of 2–11. All particles had a high negative charge, which indicated good stability of the dispersion in the alkali range. The stability of their colloidal dispersion was observed under increasing concentrations of mono- and divalent cations, and electrostatic repulsion was identified as the main stabilization mechanism. Finally, QCM-D was used to study the interaction of the lignin particles with the mineral surfaces of chalcopyrite, pyrite, and galena, which gave insight into the possible mechanism during the flotation process.