Refining best management practices (BMPs) for future highway construction depends on a comprehensive understanding of environmental impacts from current construction methods. Based on a before-after-control impact (BACI) experimental design, long-term stream monitoring (1997–2006) was conducted at upstream (as control, n = 3) and downstream (as impact, n = 6) sites in the Lost River watershed of the Mid-Atlantic Highlands region, West Virginia. Monitoring data were analyzed to assess impacts of during and after highway construction on 15 water quality parameters and macroinvertebrate condition using the West Virginia stream condition index (WVSCI). Principal components analysis (PCA) identified regional primary water quality variances, and paired t tests and time series analysis detected seven highway construction-impacted water quality parameters which were mainly associated with the second principal component. In particular, impacts on turbidity, total suspended solids, and total iron during construction, impacts on chloride and sulfate during and after construction, and impacts on acidity and nitrate after construction were observed at the downstream sites. The construction had statistically significant impacts on macroinvertebrate index scores (i.e., WVSCI) after construction, but did not change the overall good biological condition. Implementing BMPs that address those construction-impacted water quality parameters can be an effective mitigation strategy for future highway construction in this highlands region.