The expansion of Interstate 26 from Erwin, TN to the North Carolina border was a project that potentially adversely impacted South Indian Creek because of the steep landscapes and potential for erosion. Several studies have shown the short-term, negative effects of road construction on the water quality of nearby water bodies. Non-point source pollution is the major source of water pollution in the United States. The primary objective of this research is to evaluate the long-term effects of the construction of Interstate 26 on South Indian Creek to see if there has been any ecological recovery. The Environmental Health Sciences Laboratory of East Tennessee State University was contracted by the Tennessee Department of Transportation to collect data from before construction (1991-1992), during construction (1993-1994), and postconstruction (1995-1996). Comparison of microbial enzyme activities and other parameters to present-day (2012-2013) water quality conditions indicate that South Indian Creek has not fully recovered from the effects of the construction of the interstate.