This data release contains information on macroinvertebrate communities and sediment toxicity in the Niagara River and adjacent areas collected during three separate sampling efforts conducted during 2019-2020, 2022, and 2024-2025. During the first sampling effort, bed sediments were collected at 60 sites in the Niagara River, 5 sites on lower Smoke Creek, and 6 sites on Hoyt Lake for use in sediment toxicity testing and determination of grain size distribution and total organic carbon concentration. Additionally, macroinvertebrate samples were collected from the 60 sites on the Niagara River. During the second sampling effort, bed sediments were collected at 10 sites on lower Smoke Creek (5 of which were repeated from the first sampling effort), 5 reference sites on upper Smoke Creek, and 6 sites on Hoyt Lake (all of which were repeated from the first sampling effort) for toxicity testing, macroinvertebrate community assessment, and determination of grain size distribution and total organic carbon concentration. Additionally, sediments were collected at 10 sites in the Black Rock Canal, 5 reference sites in the upper Black Rock Canal, and 5 sites in Erie Basin Marina for macroinvertebrate community assessment. During the third sampling effort, bed sediments were collected at 14 sites on Cayuga Creek, 10 sites on Two Mile Creek, 8 sites on Bergholtz Creek, 7 sites on Gill Creek, 5 sites on Scajaquada Creek, 5 sites on an unnamed tributary to Bergholtz Creek, 1 site on Black Creek, and 5 reference sites on Ellicott Creek. The selection of all site locations and analyses was done by the U.S. Army Corps of Engineers and the New York State Department of Environmental Conservation based on existing data gaps and prior sediment chemistry data. For all sampling efforts, in situ habitat measurements were taken at the time of sample collection at all sites. All sediment samples were collected using a petite Ponar dredge. Ten-day sediment toxicity tests were performed using two test species, Chironomus dilutus and Hyalella azteca, following standard USEPA test methods. Macroinvertebrates were identified to the lowest practical taxonomic resolution and used to calculate metrics of biological integrity following standard New York State Department of Environmental Conservation procedures. A morphological deformity analysis was also conducted on the mentum mouthpart of midge larvae in the genus Chironomus as an additional measure of toxicity following standard New York State Department of Environmental Conservation procedures. The data release has five separate tables: one containing site locations and habitat information, one containing the results of sediment toxicity tests, one containing macroinvertebrate identifications, one containing standard New York State Department of Environmental Conservation metrics of macroinvertebrate community integrity calculated from the macroinvertebrate identifications, and one containing the results of the morphological deformity analysis.