Surface water, pore water, pond bottom sediments, and groundwater were sampled within and downgradient from five groundwater flow-through ponds that ranged from oligotrophic to eutrophic in Cape Cod, Massachusetts during different seasons from 2015 – 2018. The sampled ponds included Ashumet, Santuit, Snake, Shubael, and Longs. Pore water was collected between 15 to 100 cm below the pond bottoms on the upgradient and downgradient sides of the ponds. Groundwater was collected on the downgradient side where the pond water recharges the groundwater resulting in a “pond shadow”. Within the pond shadow, groundwater is under the direct influence of surface water. The pond shadows were sampled vertically and at various distances from their respective surface water sources. This set of data provides the microbial water quality data derived from the collection of DNA from this set of water and sediment samples. Data includes concentrations of DNA, N-cycling gene abundance for bacterial amoA, narG, nirK, and nosZ, taxonomy, and alpha and beta diversity from the amplification of 16S rRNA.