Bio-oil obtained via the vacuum pyrolysis of softwood bark residues is a multiphase, viscous, unstable system composed of water-soluble and water-insoluble fractions. Using centrifugation, the bio-oil can be separated into an upper layer (ca. 16 wt %) and a bottom layer (ca. 84 wt %). The upper layer exhibits low contents of water, solid, and ash, as well as a low density, a low acidity, a high content of methanol-insoluble materials, a high viscosity, and a high calorific value. The physicochemical properties of the bottom layer are similar to those of the whole bio-oil, except the bottom layer contains greater contents of ash and water. Microscopic analysis of the bio-oil has revealed a multiphase complex colloidal system that is composed of solid particles, three-dimensional structures, and droplets. The upper layer represents the dispersed droplet phase, which is rich in waxy materials (fatty and resin acids) and water-insoluble compounds. It is the upper layer that, overall, provides the unique colloidal properties to this type of bio-oil. The morphology of the bottom layer is similar to that of the bio-oil matrix. The presence of microstructures in the whole bio-oil has been revealed by differential scanning calorimetry and rheology. The microstructures (e.g., waxy materials) in the bio-oil matrix are, in part, responsible for the bio-oil high viscosity and non-Newtonian behavior that is observed at low temperature (<50 degreesC). This behavior disappears when the heating temperature reaches the melting point of the three-dimensional structures contained in the bio-oil. The results have confirmed the colloidal nature of the bio-oil investigated.