The interest in biomass as a source of renewable energy and chemicals has been increasing in keeping up with the transition to a sustainable bio-based economy. An important initial step of chemicals recovery from biomass-derived pyrolysis oil is water extraction where most of polar compounds are isolated in the aqueous phase. This study was done to investigate the effects of stirring rate and water-to-oil ratio on the extraction capability (distribution coefficient and yield), water content, and atomic composition of both aqueous and organic phases. The results show that the stirring rate above 300 rpm has no influence on the equilibrium. Increasing the water-to-oil ratio dilutes the aqueous phase without changing the atomic distribution. Forest residue-derived pyrolysis oil should be extracted at a water-to-oil ratio of 0.65–0.7, whereas pine-derived pyrolysis oil is preferably extracted at the lowest feasible water-to-oil ratio where complete phase separation occurs, which is 0.5 in this study.