When introduced into the aquatic environment, TiO₂ NP are likely to settle from the water column, which results in increased exposure of benthic communities. Here, we show that the activity of two extracellular enzymes of intact heterotrophic biofilms, β-glucosidase (carbon-cycling) and l-leucin aminopeptidase (nitrogen-cycling), was reduced following exposure to surface functionalized TiO₂ NP and UV radiation, depending on the particles’ coating. This reduction was partially linked to ROS production. Alkaline phosphatase (phosphorus-cycling) activity was not affected, however in contrast, an alkaline phosphatase isolated from E. coli was strongly inhibited at lower concentrations of TiO₂ NP than the intact biofilms. These results indicate that enzymes present in the biofilm matrix are partly protected against exposure to TiO₂ NP and UV radiation. Impairment of extracellular enzymes which mediate the uptake of nutrients from water may affect ecosystem function.