Biomass is one the most abundant renewable energy sources, and it can be processed through different thermochemical methods to obtain oils that can replace the petroleum bitumen used in road construction. For the construction industry to accept the bitumen replacement with bio-oil, it is necessary to know its properties and determine the applicability of conventional testing methods. This research utilized a liquified wood heavy fraction (bio-oil) obtained from waste wood through an innovative thermochemical liquefaction process. The aim was to investigate a kind of bio-bitumen produced by blending this bio-oil with paving-grade bitumen. The rheological behaviour in a wide temperature range, the performance relative to fatigue cracking and permanent deformation sensitivity, and the evolution with oxidative ageing were evaluated for the bio-bitumen and paving-grade bitumens. The bio-oil significantly affected the rheological behaviour of bitumen through an overall decrease in the phase angle and by failing the time–temperature superposition principle. The strong elastic response of the bio-bitumen improved resistance to fatigue and permanent deformation accumulation; however, resistance to oxidative ageing declined. Linear viscoelastic rheological indicators proposed in the literature to assess the material’s performance showed a similar trend of variation with oxidative ageing for bio-bitumen and paving-grade bitumen, though the indicators’ values could not be directly compared.