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Understanding mixed-land-use practices and physicochemical influences on Escherichia (E.) coli concentrations is necessary to improve water quality management and human health. Weekly stream water samples and physicochemical data were collected from 22 stream gauging sites representing varying land use practices in a contemporary Appalachian watershed of the eastern USA. Over the period of one annual year, Escherichia (E.) coli colony forming units (CFU) per 100 mL were compared to physicochemical parameters and land use practices. Annual average E. coli concentration increased by approximately 112% from acid mine drainage (AMD) impacted headwaters to the lower reaches of the watershed (approximate averages of 177 CFU per 100 mL vs. 376 CFU per 100 mL, respectively). Significant Spearman’s correlations (p &lt; 0.05) were identified from analyses of pH and E. coli concentration data representing 77% of sample sites; thus highlighting legacy effects of historic mining (AMD) on microbial water quality. A tipping point of 25–30% mixed development was identified as leading to significant (p &lt; 0.05) negative correlations between chloride and E. coli concentrations. Study results advance understanding of land use and physicochemical impacts on fecal contamination in mixed-land-use watersheds, aiding in the implementation of effective water quality management practices and policies.

Advancing Understanding of Land Use and Physicochemical Impacts on Fecal Contamination in Mixed-Land-Use Watersheds