The Upper Esopus Creek receives water diversions from a neighboring basin through the Shandaken Tunnel (the portal) from the Schoharie Reservoir. Although the portal is closed during floods, mean flows and turbidity of portal waters are generally greater than in Esopus Creek above their confluence. These conditions could potentially affect local fish assemblages, yet such effects have not been assessed in this highly regulated stream. We studied water quality, hydrology, temperature, and fish assemblages at 18 sites in the Upper Esopus Creek during 2009–2011 to characterize the effects of the portal input on resident-fish assemblages and to document the status of the fishery resource. In general, fish-community richness increased by 2–3 species at mainstem sites near the portal, and median density and biomass of fish communities at sites downstream of the portal were significantly lower than they were at sites upstream of the portal. Median densities of Salmo trutta (Brown Trout) and all trout species were significantly lower than at mainstem sites downstream from the portal—25.1 fish/0.1 ha and 148.9 fish/0.1 ha, respectively—than at mainstem sites upstream from the portal—68.8 fish/0.1 ha and 357.7 fish/0.1 ha, respectively—yet median biomass for Brown Trout and all trout did not differ between sites from both reaches. The median density of young-of-year Brown Trout at downstream sites (9.3 fish/0.1 ha) was significantly lower than at upstream sites (33.9 fish/0.1 ha). Waters from the portal appeared to adversely affect the density and biomass of young-of-year Brown Trout, but lower temperatures and increased flows also improved habitat quality for mature trout at downstream sites during summer. These findings, and those from companion studies, indicate that moderately turbid waters from the portal had few if any adverse impacts on trout populations and overall fish communities in the Upper Esopus Creek during this study.