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The mechanisms involved in N2O production, consumption and air-sea interactions in fluvial systems are modulated and affected by several factors, including hydrological, chemical and anthropogenic impacts. In recent decades, the basin of the Biobío River (central Chile) has been impacted by an enhanced precipitation deficit and anthropogenic pressure, so the aim of the present work is to understand the spatial and seasonal dynamics of N2O during a megadrought that has been affecting the river since 2010. We also aim to determine the annual contribution of N2O to the atmosphere and the possible response of the Biobío River to projected climatic changes. Seasonal sampling of the water physical-chemical properties was carried out at 15 stations along 280 km of the river and its five tributaries; the stations were distributed between the pristine headwaters (700 m asl) and the outlet of the river at the Pacific Ocean. The longitudinal distribution of oxygen, nutrients, and nitrous oxide evidenced agricultural, urban and industrial impacts on the river water. Headwater areas presented the highest oxygen concentration and the lowest nutrient and N2O concentrations, all of which fluctuated with the variability in water discharge with the different seasons. In the middle river section, where agricultural, industrial and urban activities impacted the river, the nutrient and N2O concentrations increased up to 20 and 1.2 times, respectively, compared to those in the headwaters, and the outlet area showed the highest nutrient and N2O concentrations. Throughout the entire river, N2O oversaturation exhibited a pronounced seasonal cycle with maxima occurring during the dry season. Our results suggest that urban activities had the greatest impact on the Biobío River at its outlet. Furthermore, the inverse relationship between the N2O concentration and water discharge suggests that the predicted future decrease in water discharge may result in higher N2O values in the Biobío River that would expectedly enhance global warming further, through a positive feedback.