Energy development threatens fish and wildlife resources worldwide. This study used constrained ordinations to show fish assemblage structure associated with oil and gas well densities in the Colorado River Basin, Wyoming, but well densities explained only 6.4% of assemblage structure when compared to other factors. Threshold Indicator Taxonomic ANalysis showed significant negative threshold responses by some species to small levels of development (<0.15 wells km−2), whereas positive thresholds were less distinct. Some native and imperilled species could be disproportionately affected if future oil and gas development proceeds in a manner that will impact aquatic resources. Although existing development has not substantially influenced regional fish assemblage structure, it appears to affect a subset of species. Understanding assemblage-level responses to development can help land managers determine appropriate development levels, prioritise areas for monitoring associated with future development and identify where land protection measures may be needed to offset potential risks.