Methylmercury (MeHg), a neurotoxic pollutant, is formed mainly under anaerobiosis. The “Modulo Sperimentale Elettromeccanico” (MOSE), built to temporarily close the Venice Lagoon and protect the city from flooding, induces changes in the hydrological regime, reducing water circulation and decreasing in the dissolved oxygen concentrations of the lagoon. Our study shows the potential changes in sediment and overlying water physicochemistry in a simulated MOSE closing-event by incubating sediment cores for 48 h in the laboratory and deploying benthic chambers. In the incubated summer cores (September 2021), a significant increase in total Hg and MeHg concentrations in the water column was observed and associated with an increase in MeHg formation rates – particularly, MeHg formation rates doubled during the simulated MOSE-closing. This increase was associated to a release of dissolved organic carbon and to an enrichment of proteinaceous substances and reactive humic acids in the overlying waters. All these effects were not evident in late autumn (November 2019), when water temperature was 10 ◦C lower than in September 2021. Our study suggests that hydrological changes caused by the MOSE closure may in some periods increase MeHg concentrations within the Venice Lagoon.