In North America, one of the primary sources of stress on aquatic systems is agricultural land use. Streams are nested, hierarchical structures wherein the larger scale characteristics constrain the smaller components, determining instream ecology. The objective of this study was to assess the influence of different landscape and instream characteristics on water quality by assessing watershed characteristics, water chemistry and benthic community structure across a gradient of agricultural intensity in the Grand River watershed in Southern Ontario, Canada. Twenty-one sites transecting the central watershed were sampled in October 2020, and June 2021. Results of a Canonical Correspondence Analysis and structural metrics showed significant differences between microbasins across the watershed based on underlying natural and anthropogenic factors. Lower pollution tolerance values were found to be associated with micro-basins that had lower agricultural extent, less artificial tile drainage and greater riparian buffer presence, suggesting that land use and buffer extent play a key role in determining stream health.