This study was conducted in the Talladega research watershed (33.76219799, -85.59550775), along an unnamed intermittent tributary of Pendergrass Creek, in the Talladega National Forest within the Piedmont Upland physiographic province (Al, USA). The creek's drainage area spans an elevation range of 150–730 m and contributes to Choccolocco Creek, a tributary of the Coosa River. The region experiences a humid subtropical climate, with mean annual temperatures ranging from -2°C to 32°C and annual precipitation of 140–160 cm. The surrounding vegetation includes a mix of deciduous forests and pine plantations, while the streambed comprises saprolite cobble, gravel, and sand substrates. This synoptic survey was designed in support of the sampling goals of the Aquatic Intermittency effects on Microbiomes in Streams (AIMS) Project. In June 2022, a field team collected datasets characterizing hydrology, biogeochemistry, and ecology at 48 locations within a sub-drainage of Pendergrass Creek. Sites were selected to balance multiple competing priorities: (i) strategically targeting existing monitoring infrastructure with long-term data; (ii) including sites near known springs and tributary junctions; and (iii) representing a range of drainage area and topographic wetness index (TWI) values, both of which have been correlated with flow permanence. Briefly, the sites selected based on drainage area and TWI were chosen by binning drainage area into 10 bins and then binning TWI into quintiles within each drainage area bin. We then randomly selected a point in each bin after accounting for points selected based on existing infrastructure, springs, and tributaries; and enforcing a minimum spacing of 100 m between locations. We then made minor adjustments to points to account for field conditions, for instance adjusting locations with respect to a road crossing. At each location, samples of water (if present), biofilm, leaf litter, and sediment were collected. The activity of microbial enzymes associated with organic matter decomposition (β-glucosidase, phenol oxidase, and peroxidase) and the mineralization of organic nitrogen (N-acetylglucosaminidase) and phosphorus (phosphatase) were measured. Enzyme activities were quantified fluorometrically using 4-methylumbelliferyl (MUB)-linked substrates or colorimetrically using 4-nitrophenyl (pNP)-linked substrates and 3,4-dihydroxyphenylalanine (L-DOPA)-linked substrates.