Due to climate change, the frequency and duration of flood events are predicted to increase in many regions of the world. This is expected to cause large changes in soil functioning and to a progressive decline in soil quality such as reduced rates of nutrient cycling, enhanced greenhouse gas emissions and loss of soil biodiversity. There is a knowledge gap, however, on how temperate agricultural soils under different management practices (e.g. manure application) respond to prolonged river or coastal flooding. The main objective of this work was to determine the effects of a simulated prolonged flooding with saline and freshwater on soil N cycling, following application of a low C:N organic amendment (broiler litter) at two temperatures, representative of a winter and a spring flood event. Using laboratory mesocosms we simulated prolonged winter (6 °C) and spring (14 °C) flooding of soil amended with broiler litter. We also compared the effects of inundation with either river (freshwater) or coastal (saline) water. An agricultural grassland soil (Eutric Cambisol) was subjected to different combinations of treatments (flood with fresh or saline water, winter vs spring temperatures, with/without poultry manure). The impact of these treatments on soil solution N dynamics, greenhouse gas emissions (CO2, CH4, N2O) and microbial community structure (by PLFA analysis) were evaluated over an 11 week simulated flood event followed by an 8 week soil recovery period (without flood). Overall, potential losses of NH4 + and cumulative GHG emissions were increased by flooding and the presence of manure. CH4 emissions were found to dominate under freshwater flooding conditions and N2O under saline flooding. Significant releases of GHG occurred during both flooding and after floodwater removal. Temperature was less influential on regulating GHG under the different treatments. These releases in GHG were associated with disruption in N cycling and changes in soil microbial composition and these changes persisted after floodwater removal. Extreme flooding negatively impacts soil functioning, however, the magnitude of any changes remain critically dependent on flood 3 duration and source of flood water, and management conditions. Further work is required at the field scale to understand the molecular basis of the responses observed in this study. Highlights • NH4 + losses and GHG emissions were increased by flooding in the presence of manure. • CH4 is mostly emitted in freshwater flooding and N2O in saline flooding. • Temperature was less important in regulating GHG emissions but did affect N cycling under flooding. • Flooding induces prolonged shifts in the composition of the soil microbial community. • Extreme flooding events negatively impact soil functioning.