<p>Microbial communities in streams occupy diverse habitats and drive nutrient cycling and organic matter dynamics. However, streams are impacted by climate change and human activities, with many small streams only flowing intermittently. Spatial patterns in microbial activity in these systems are poorly understood, especially in co-occurring habitats. This study investigated how microbial activity varied across four different habitats in an intermittent stream network. The activity of enzymes involved in organic matter decomposition (β-glucosidase, phenol oxidase, peroxidase) and organic phosphorus (phosphatase) and nitrogen (N-acetylglucosaminidase) mineralization was determined for water, epilithic biofilm, leaf litter and sediment, collected from 48 locations across 1.3 km² in Kings Creek, Kansas, USA. Microbial activity varied in magnitude and spatial distribution across the stream network, with spatial patterns differing by enzyme and habitat. Activities of β-glucosidase, phosphatase, and N-acetylglucosaminidase were correlated across all habitats, and positively correlated with phenol oxidase and peroxidase activity in leaf litter and sediment. Spatial patterns in activity were influenced by several factors, with moisture content, drainage area and topographic wetness index showing contrasting effects across habitats. Enzyme stoichiometry suggested that microbial communities in water and sediment were mainly co-limited by carbon and phosphorus, while those in biofilms were primarily nitrogen-limited, and those in leaf litter were either limited by nitrogen or co-limited by carbon and phosphorus. This study shows pronounced spatial heterogeneity in microbial activity within a small catchment, emphasizing how interactions between reach-scale and watershed-scale factors regulate organic matter and water availability, thereby shaping microbial function.</p>