(1) Background: This paper discusses the impact of agricultural activities on stream health, particularly in relation to dairy cow fecal pollution. The study explores the fecal microbiome of cattle and the potential ecological implications of aging fecal pollution on waterways. (2) Methods: The study examines changes in the bacterial community available for mobilization from in-situ decomposing cowpats and the effects of simulated rainfall. The microbiome of individual cowpats was monitored over 5.5 months. We used 16S rRNA metagenomics and machine learning software, FEAST (Fast Expectation-mAximization for microbial Source Tracking), for bacterial and fecal source assignments. (3) Results: The phyla Bacillota and Bacteroidota are dominant in the fecal microbiota of fresh cow feces but shift to Pseudomonodota, Actinomycetota, and environmental Bacteroidota in aged cowpats. Potential impacts of these bacterial community shifts on inputs to local agricultural streams are discussed in relation to water quality monitoring and aging sources of fecal contamination. We identified taxon orders that are potential indicators of fresh cattle sources (Oscillospirales and Bacteroidales) and aged sources (Peptostreptococcales-Tissierellales) in water bodies. (4) The paper highlights that bacterial metagenomic profiling can inform our understanding of the ecology of microbial communities in aquatic environments and the potential impacts of agricultural activities on ecosystem health.