Abstract. Nitrous oxide (N₂O) is a potent greenhouse gas, generated through microbial nitrogen (N) turnover processes, such as nitrification, nitrifier denitrification, and denitrification. Previous studies quantifying natural sources have mainly focused on soils and the ocean, but the potential role of terrestrial water bodies in the global N₂O budget has been widely neglected. Furthermore, the biogeochemical controls on the production rates and the microbial pathways that produce benthic N₂O in lakes are essentially unknown. In this study, benthic N₂O fluxes and the contributions of the microbial pathways that produce N₂O were assessed using ¹⁵N label flow-through sediment incubations in the eutrophic, monomictic south basin of Lake Lugano in Switzerland. The sediments were a significant source of N₂O throughout the year, with production rates ranging between 140 and 2605 nmol N₂O h⁻¹ m⁻², and the highest observed rates coinciding with periods of water column stratification and stably anoxic conditions in the overlying bottom water. Nitrate (NO₃⁻) reduction via denitrification was found to be the major N₂O production pathway in the sediments under both oxygen-depleted and oxygen-replete conditions in the overlying water, while ammonium oxidation did not contribute significantly to the benthic N₂O flux. A marked portion (up to 15%) of the total NO₃⁻ consumed by denitrification was reduced only to N₂O, without complete denitrification to N₂. These fluxes were highest when the bottom water had stabilized to a low-oxygen state, in contrast with the notion that stable anoxia is particularly conducive to complete denitrification without accumulation of N₂O. This study provides evidence that lake sediments are a significant source of N₂O to the overlying water and may produce large N₂O fluxes to the atmosphere during seasonal mixing events.