Introduced rainbow trout Oncorhynchus mykiss and brown trout Salmo trutta fario are the most abundant fishes in the northern Chilean Patagonia, and their effect on native fishes is not well known. We tested for interactive segregation between trout and native fishes by using a before-after, control-impact design in which we deliberately reduced the density of trout and observed the response of the native fishes in their mesohabitat use (pool, run, riffle). Three native fish species, Brachygalaxias bullocki, Galaxias maculatus (inanga), and Trichomycterus areolatus, apparently had niche overlap with introduced trout and changed their mesohabitat use after trout reduction. The expansion of the three species into a wider range of mesohabitats after trout reduction suggests that these fishes occupy a broader spatial area when trout are reduced or possibly absent. However, some native fish species, such as Geotria australis, did not respond to the trout reductions. To protect Chilean native fishes, policymakers need to consider providing legal protection for native fishes because native fishes currently have no protection in their catchments.