Fast pyrolysis of biomass is a well-known opportunity for sustainable alternative fuel production for transport and energy. However, bio-oils from biomass pyrolysis are viscous, acidic bio-crudes that need further steps of upgrading before being used either as fuels or chemicals. A process that is complementary to bio-oil hydrotreatment or co-processing consists of optimizing and tuning the upstream condensation steps of fast pyrolysis to separate and concentrate selected classes of compounds. This can be implemented by varying the condensation temperatures in a multi-step condensation unit. In this study, fractional condensation of fast pyrolysis vapors from pinewood has been applied to a bubbling fluidized bed reactor of 1 kg h−1 feed. The reactor was operated at 500 °C and connected to a downstream interchangeable condensation unit. Tests were performed using two different condensing layouts: (1) a series of two spray condensers and a tube-in-tube water-jacketed condenser, referred to as an intensive cooler; (2) an electrostatic precipitator and the intensive cooler. Using the first configuration, which is the focus of this study, high boiling point compounds—such as sugars and lignin-derived oligomers—were condensed at higher temperatures in the first stage (100–170 °C), while water-soluble lighter compounds and most of the water was condensed at lower temperatures and thus largely removed from the bio-oil. In the first two condensing stages, the bio-oil water content remained below 7% in mass (and therefore, the oil’s high calorific content reached 22 MJ kg−1) while achieving about 43% liquid yield, compared to 55% from the single-step condensation runs. Results were finally elaborated to perform a preliminary energy assessment of the whole system toward the potential upscaling of this fractional condensation approach. The proposed layout showed a significant potential for the upstream condensation step, simplifying the downstream upgrading stages for alternative fuel production from fast pyrolysis bio-oil.