The fate and the methylation of mercury (Hg) in the terrestrial environment are still poorly understood and although the main drivers of release and methylation of mercury in soils are known (low redox potential and microbial carbon availability) their interactions are not well understood. This is of concern since many agriculturally used floodplains, where the recurring flooding and agricultural practices (e.g. manure amendments) may have an impact on the fate and the biomethylation of Hg, are at the same time Hg-contaminated. In this study, we modified and validated existing methods to extract and analyze methylmercury (MeHg) by HPLC-ICP-MS in soils and we assessed the Hg and MeHg concentrations in three fields situated in a Hg polluted agricultural floodplain. Further, we incubated the top soil from the three studied fields for 11 days under flooded conditions in presence or absence of 2 mass% of cow manure, a common agricultural amendment in the area. Total Hg and MeHg concentrations ranged from <limit of detection (LOD, 0.012 mg kg⁻¹) to 28.2 mg kg⁻¹ and from 1.2 to 7.8 μg kg⁻¹ respectively. Hg was released to the soil solution after 12 hours with a maximum between day 2 and day 7. MeHg levels in the soil solution were <LOD although it was found in the soil before and after the incubation. The addition of cow manure to saturated soils led to an increase in the MeHg concentrations of the soil solid phase by up to fivefold to a maximum of 26.4 ± 1.1 μg kg⁻¹ (n = 3). Our study demonstrates that the combination of low redox potential because of flooding with common agricultural practices such as the amendment of manures enhances the formation of toxic MeHg.