Agricultural conservation practices, widely implemented across the U.S., are intended to protect stream health from deleterious effects of agriculture. Biotic assemblages are the most integrative indicators of stream health but weak biotic responses commonly indicate that conservation practices are less effective than expected. Moreover, despite billions spent to implement conservation practices, health of streams draining agricultural lands continues to decline across the U.S. We sought to quantify detectable biophysical effects of conservation practices in watersheds where pastureland is a predominant land use. We collected data on water quality, instream habitat conditions, and macroinvertebrate assemblages from 31 sites in the upper Tennessee River basin of southwest Virginia, U.S. We also collated conservation practice and land cover data for watersheds surrounding those sites. Several statistical methods were used to examine linear and nonlinear relationships among water quality, instream habitat, macroinvertebrate assemblage composition, land cover, and conservation practice density. Our results confirmed that the mitigating effects of conservation practices on agriculture’s impacts on stream health follow complex paths and are difficult to detect. Few bivariate relations between physicochemical and biotic metrics provided evidence of conservation practice efficacy. However, we did find watershed-level density of practices to be positively correlated with site-level bank stability, and an apparent threshold of practice density associated with effects on site-level total nitrogen. We also observed many positive and negative threshold responses by macroinvertebrates to physicochemical conditions, but these thresholds commonly occurred at levels below the conditions associated with current implementation patterns (i.e., types and densities) of conservation practices. Further, the suite of taxa exhibiting threshold responses varied widely among specific physicochemical gradients. We conclude that conservation practices can influence stream biota via effects on water and habitat quality, but current implementation densities, durations, and/or specific placements of practices are inadequate to restore or maintain healthy biotic assemblages.