Headwaters can represent 80% of stream kilometers in a watershed, and they also have unique physical and biological properties that have only recently been recognized for their importance in sustaining healthy functioning stream networks and their ecological services. We sampled 60 headwater tributaries in the South Fork Trinity River, a 2,430 km2, mostly forested, multiple-use watershed in northwestern California. Our objectives were: (1) to differentiate unique headwater types using 69 abiotic and vegetation variables measured at three spatial scales, and then to reduce these to informative subsets; (2) determine if distinct biota occupied the different tributary types; (3) determine the environmental attributes associated with the presence and abundance of these biotic assemblages; and (4) using niche modeling, determine key attribute thresholds to illustrate how these biota could be employed as metrics of system integrity and ecological services. Several taxa were sufficiently abundant and widespread to use as bio-indicators: the presence and abundance of steelhead trout (Oncorhynchus mykiss), herpetofauna (reptile and amphibian) species richness, and signal crayfish (Pacifastacus leniusculus) represented different trophic positions, value as commercial resources (steelhead), sensitivity to environmental stress (amphibians), and indicators of biodiversity (herpetofauna species richness). Herpetofauna species richness did not differ, but abundances of steelhead trout, signal crayfish, and amphibian richness all differed significantly among tributary types. Niche models indicated that distribution and abundance patterns in both riparian and aquatic environments were associated with physical and structural attributes at multiple spatial scales, both within and around reaches. The bio-indicators responded to unique sets of attributes, reflecting the high environmental heterogeneity in headwater tributaries across this large watershed. These niche attributes represented a wide range of headwater environments, indicating responses to a number of natural and anthropogenic conditions, and demonstrated the value of using a suite of bio-indicators to elucidate watershed conditions, and to examine numerous disturbances that may influence ecological integrity.