This U.S. Geological Survey (USGS) data release is an assessment of aquatic habitat within the tributaries of the lower Middle Fork Salmon River. These datasets are primarily focused on physical stream habitat (2020-21 stream temperature data) and the presence or absence (2021 eDNA data) of fish that inhabit the basin. Many environmental stressors contribute to the declining range and density of Bull trout (S. confluentus) populations, one of which is the toxic trace metals contributions from legacy mine waste and adits. These data were collected in stream habitats with variable physical and thermal conditions. Characterizing and comparing reference streams of similar physical attributes will assist to differentiate the regional impacts of toxic trace metals exposure.