A data release containing information on macroinvertebrate communities and sediment toxicity in the Niagara River and associated areas collected during 2019 and 2020. Sediment toxicity tests were conducted at 60 sites in the Niagara River, 5 sites on Smoke Creek, and 6 sites on Hoyt Lake using two test species, Chironomus dilutus and Hyalella azteca, following USEPA test methods 100.2 and 100.1, respectively. In situ habitat measurements and sediment samples for determination of grain size distribution and total organic carbon concentration were also taken at the time of sample collection at all sites. Additionally, macroinvertebrate samples were collected using a petite Ponar dredge from the same 60 sites on the Niagara River. 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 all 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 10-day 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.