AbstractStream microbial communities and associated processes are influenced by environmental fluctuations that may ultimately dictate nutrient export. Discharge fluctuations caused by intermittent stream flow are increasing worldwide in response to global change. We examined the impact of flow cessation and drying on in‐stream nitrogen cycling. We determined archaeal (AOA) and bacterial ammonia oxidizer (AOB) abundance and ammonia oxidation activity in surface and deep sediments from different sites along the Fuirosos stream (Spain) subjected to contrasting hydrological conditions (i.e., running water, isolated pools, and dry streambeds). AOA were more abundant than AOB, with no major changes across hydrological conditions or sediment layers. However, ammonia oxidation activity and sediment nitrate content increased with the degree of stream drying, especially in surface sediments. Upscaling of our results shows that ammonia oxidation in dry streambeds can contribute considerably (~50%) to the high nitrate export typically observed in intermittent streams during first‐flush events following flow reconnection. Our study illustrates how the dry channels of intermittent streams can be potential hotspots of ammonia oxidation. Consequently, shifts in the duration, spatial extent and severity of intermittent flow can play a decisive role in shaping nitrogen cycling and export along fluvial networks in response to global change.