<p>Natural ponds and constructed reservoirs (dugouts) provide essential water for agriculture, yet the effects of livestock disturbances on the freshwater microbial ecosystems remain underexplored. In this study, 16S rRNA gene sequencing was combined with water geochemistry analyses to examine the relative influence of livestock and geochemical variables on microbial communities in the Prairie Pothole Region (PPR) of the North American Great Plains. In undisturbed ponds, geochemical factors such as dissolved organic carbon (DOC) and sulfate (SO₄²⁻) create strong selective pressures, creating distinct microbial communities specialized in carbon and sulfur cycling, leading to niche differentiation and the dominance of specific bacterial taxa. In contrast, livestock-induced disturbances like manure deposition and physical disruption increase within-pond spatial heterogeneity, weakening local geochemical control, while simultaneously reducing biodiversity and homogenizing communities across ponds despite underlying geochemical differences. One example of cattle-induced disturbances was their impact on relative abundances of multiple microorganisms such as <i>Thiobacillus. Thiobacillus</i> is a major contributor to sulfur cycling, converting hydrogen sulfide (H₂S) into SO₄²⁻ under aerobic conditions and contributing to sulfur sequestration by incorporating SO₄²⁻ into biomass through assimilatory SO₄²⁻ reduction. In ponds with cattle activities <i>Thiobacillus</i> relative abundance was decreased and the impact of SO₄²⁻ and DOC was less prominent. Overall, this study showed that livestock disturbances disrupt local microbial community assembly and weaken the influence of geochemical selection in structuring pond-scale microbial diversity.</p>