Non-native trout (Oncorhynchus mykiss, Salmo trutta) invasions have been implicated in the decline of native galaxiid fishes throughout temperate Southern Hemisphere freshwaters. As many Galaxias fishes are endangered, knowledge of factors facilitating localised co-occurrence of native and introduced species is needed for both conservation and management. While recent research demonstrates a role for flow-related mediation of trout effects on galaxiids, the mechanism(s) underlying such phenomena remain unclear. Accordingly, we assessed the interplay between environmental conditions and trophic niches for both species across a gradient of co-occurrence and hydrology. In particular, we quantified the trophic position (TP), energy sourcing patterns, and niche breadth based on the abundance of naturally occurring stable isotopes (15N and 13C) within muscle tissues for trout and galaxiids in replicate food webs from streams varying widely in size and disturbance regime. We found that both species held similar trophic positions, but TP varied considerably across individuals and sites due to combined effects of body size and hydrological disturbance. Further, patterns in δ13C revealed an effect of disturbance on the relative reliance by fish on a key prey taxon, and a ca. 1 ‰ shift in δ13C by galaxiids in the presence of trout. Lastly, isotope-based measures of niche width, which were positively correlated with coarse dietary measures of niche width, were similar, albeit variable, for all groups. Galaxiid niche width co-varied negatively with relative trout abundance, whereas that for trout did so with stream size. Altogether, our findings demonstrate that both the presence of trout and environmental conditions act in concert to influence where galaxiids are positioned within stream food webs. Perhaps more importantly, they suggest that flexible niches may enable galaxiids to persist in the presence of a species that might otherwise cause their local extinction.