Premature failure at the bitumen−aggregate interface is attributed to the surface chemistry of stone aggregates. Bitumen modifiers with high affinity toward aggregates are often used to change the surface chemistry of aggregates, which in turn changes the type and strength of stones' interactions with bitumen.Here, the mechanisms of action and the effects of an organosilane and bio-oil separately on the bitumen−aggregate interface, including their binding to aggregates from one side and to bitumen from the other side, were studied and compared. The binding of bio-oils and organosilanes to aggregates is well documented, as well as their efficacy as anti-stripping agents, but their binding to bitumen is not well understood. Using glass beads as a surrogate for siliceous stones, we obtained laboratory measurements of interfacial strength as measured by resistance to shear forces at the interface of bitumen and silica. Our laboratory measurements showed the highest value for the bitumen modified with bio-oil, followed by the control bitumen with no modification, and the lowest value for the bitumen modified with organosilane. Our molecular modeling calculations showed that the adhesion of bitumen molecules to silica coated with bio-oil was notably stronger (up to 72%) than the adhesion of bitumen molecules to silica coated with organosilane. The higher interfacial strength observed for bitumen with bio-oil is attributed to bio-oil having various functional groups with the ability to form hydrogen bonds and π−π interactions with the aromatic and polar components of bitumen. The study outcomes highlight the importance of modifiers' ability to strongly interact with both aggregates and bitumen. The bio-oil modifier introduced in this work can form a robust bridge between bitumen and siliceous aggregates to improve the adhesive properties, enhancing the sustainability and durability of bituminous composites.