The Mount Amiata mining district (southern Tuscany, Italy) was, for decades, one of the world's largest mercury (Hg) producing regions, where mining activity lasted until the 1980s. The Paglia River drains the eastern part of the district and is also the main western tributary of the Tiber River. Recent studies show that, still today, high total Hg contents severely affect the downstream ecosystems of these rivers. In November 2012, a major flood event occurred in the Paglia River basin, which drastically changed the river morphology and, possibly, the Hg concentrations. In the present work, stream sediment was sampled before and after the flood to evaluate possible changes in sediment total Hg concentrations as a consequence of this event. The comparison between pre- and post-flood Hg concentrations shows that Hg content increased up to an order of magnitude after the flood, suggesting that this event triggered Hg mobilization in the basin rather than its dilution.