Mercury contamination is of high concern due to its bioaccumulation, toxicity and biomagnification along the food chain. Biofilms can accumulate Hg and contribute to its incorporation in freshwater food webs. Nevertheless, the accumulation kinetics of Hg by biofilms is not well described and understood. The aim of the present study was thus to gain mechanistic understanding of Hg accumulation by biofilms. Kinetics of Hg uptake by biofilms of different ages (e.g. different compositions) was characterised by determining Hg contents in biofilms with and without a cysteine-washing step. Hg accumulation was rapid in both biofilms, with the uptake rate constant of the younger biofilm 10 times higher than that of the older biofilm. Moreover, accumulated Hg reached a plateau at 24 h exposure in the younger biofilm, whereas it increased linearly in the older biofilm. The observed difference in Hg uptake by the studied biofilms is likely a result of the difference in biofilm thickness (and thus Hg diffusion inside the biofilm matrix) and microbial composition. These findings have important implications for the understanding of Hg bioavailability and effects towards aquatic microorganisms.