This study was conducted in the Talladega watershed (outlet: 33.7622, -85.5955), located in the Talladega National Forest (Cleburne County, AL) and drains a non-perennial tributary of Pendergrass Creek. Covering 0.92 km² of mixed coniferous and deciduous forest in the Piedmont Upland physiographic section, the watershed ranges in elevation from 345 to 456 m and ultimately drains into the Coosa River (Mobile-Tombigbee basin). The region has a humid subtropical climate with mean January and July temperatures of 5.3°C and 25.3°C, and annual precipitation of 1,400 mm. Paint Rock watershed (outlet: 34.9686, -86.1650) lies on private land in Jackson County, AL, near Estillfork. It drains a non-perennial tributary to Burks Creek and spans 2.97 km² of deciduous forest in the Cumberland Plateau. Elevation ranges from 211 to 550 m, and the watershed feeds into the Paint Rock River (Tennessee basin). The climate is humid subtropical, with mean January and July temperatures of 4.4°C and 25.4°C, and annual precipitation of 1,390 mm. Weyerhaeuser watershed (outlet: 32.9841, -88.0133) is on private land in Greene County, AL, near Eutaw. It drains a non-perennial tributary to Shambley Creek and includes 0.70 km² of coniferous forest managed for silviculture in the East Gulf Coastal Plain. Elevation ranges from 63 to 94 m, and the watershed contributes to the Sipsey River (Mobile-Tombigbee basin). The climate is humid subtropical, with mean January and July temperatures of 7.3°C and 27.4°C, and precipitation of 1,350 mm. These watersheds were part of a seasonal sampling survey (January 2022–January 2024) for the AIMS Project. Data on hydrology, biogeochemistry, and microbial ecology were collected at 7 sites per watershed. At each site, water (if present), biofilm, leaf litter, and sediment were sampled. Microbial enzyme activities were measured to assess organic matter decomposition (β-glucosidase, phenol oxidase, peroxidase) and nutrient mineralization (N-acetylglucosaminidase for nitrogen, phosphatase for phosphorus) using fluorometric (MUB) and colorimetric (pNP, L-DOPA) assays.