Sedimentary prokaryotes are primarily responsible for metabolic activity in rivers. They are subjected to a variety of natural and anthropogenic pressures, allowing us to use the composition and diversity of their communities as indicators of ecological health. For our investigation, we looked at sediments from three ecologically critical streams in the Lower Tallapoosa River Basin (Choctafaula, Uphapee, and Cubahatchee Creeks). The prokaryotic community was characterized using molecular approaches, and elemental concentrations were determined by inductively coupled plasma atomic emission spectrometry. This research demonstrated that Proteobacteria (45.02–80.73%), Bacteroidetes (1.98–26.52%), Firmicutes (1.36–50.67%), Actinobacteria (1.55–16.81%), and Acidobacteria (0.13–8.77%) were the most prevalent phyla. Key physicochemical parameters, core communities at multiple taxonomic levels, and several pathogenic genera shifted radically between streams. Weighted and unweighted unifrac distance metrics based PCoA plots indicated the structural and membership similarity of samples from specific creek ecosystems. Based on our research findings, it is evident that the composition and diversity of prokaryotic communities in sediment could serve as significant indicators in stream ecosystems. Further investigation and application of these indicators could prove valuable in assessing the health of streams, particularly in light of the accelerated changes resulting from climate change within a condensed timeframe.