Biofilm fouling significantly impacts ship performance. Here, the impact of biofilm on boundary layer structure at a ship-relevant, low Reynolds number was investigated. Boundary layer measurements were performed over slime-fouled plates using high resolution particle image velocimetry (PIV). The velocity profile over the biofilm showed a downward shift in the log-law region (ΔU+), resulting in an effective roughness height (ks) of 8.8 mm, significantly larger than the physical thickness of the biofilm (1.7 ± 0.5 mm) and generating more than three times as much frictional drag as the smooth-wall. The skin-friction coefficient, Cf, of the biofilm was 9.0 × 10−3 compared with 2.9 × 10−3 for the smooth wall. The biofilm also enhances turbulent kinetic energy (tke) and Reynolds shear stress, which are more heterogeneous in the streamwise direction than smooth-wall flows. This suggests that biofilms increase drag due to high levels of momentum transport, likely resulting from protruding streamers and surface compliance.