Biofilm fouling has a significant effect on ship performance. Here, the impact of biofilm fouling on boundary layer structure is investigated. Turbulent boundary layer measurements were performed over diatomaceous-slime-fouled plates using high resolution PIV. The mean velocity profile over biofilm shows the expected downward shift (ΔU+), producing higher drag, and hence higher friction velocity. This increase in drag is seen in enhanced turbulent kinetic energy and Reynolds shear stress. Due to the complex nature of the biofilm’s topography, the flow is heterogeneous in the streamwise direction when compared with smooth-wall flows.