We examined thiaminase activity in dreissenid mussels collected at different depths and seasons, and from various locations in Lakes Michigan, Ontario, and Huron. Here we present evidence that two dreissenid mussel species (Dreissena bugensis and D. polymorpha) contain thiaminase activity that is 5–100 fold greater than observed in Great Lakes fishes. Thiaminase activity in zebra mussels ranged from 10,600 to 47,900 pmol g^-1·min^-1 and activities in quagga mussels ranged from 19,500 to 223,800 pmol g^-1 ·min^-1. Activity in the mussels was greatest in spring, less in summer, and least in fall. Additionally, we observed greater thiaminase activity in dreissenid mussels collected at shallow depths compared to mussels collected at deeper depths. Dreissenids constitute a significant and previously unknown pool of thiaminase in the Great Lakes food web compared to other known sources of this thiamine (vitamin B₁)-degrading enzyme. Thiaminase in forage fish of the Great Lakes has been causally linked to thiamine deficiency in salmonines. We currently do not know whether linkages exist between thiaminase activities observed in dreissenids and the thiaminase activities in higher trophic levels of the Great Lakes food web. However, the extreme thiaminase activities observed in dreissenids from the Great Lakes may represent a serious unanticipated negative effect of these exotic species on Great Lakes ecosystems.