Benthic prey fishes are a critical component of the Lake Ontario food web, serving as energy vectors from benthic invertebrates to native and introduced piscivores. Beginning in 1978, Lake Ontario benthic prey fishes were assessed using bottom trawls collected from the lake’s south shore (depth range: 8 – 150 m). Historically, the survey targeted the then dominant species, Slimy Sculpin, however in 2015, the Benthic Prey Fish Survey was cooperatively expanded to a whole-lake survey, to address resource management information needs related to Round Goby, Deepwater Sculpin, and nearshore native fishes. In 2016, 142 trawls were collected at 18 transects, and spanned depths from 6 – 225 m. Trawl catches indicated the benthic and demersal prey fish community was dominated by Round Goby, however the proportional importance of native Deepwater Sculpin is increasing. Species-specific assessments found lake-wide Round Goby density (~600 fish per hectare) was slightly lower in 2016 relative to 2015. Deepwater Sculpin density has generally increased since 2004. In 2016 their estimated density was greater than 100 fish per hectare. Slimy Sculpin density (15 fish/ha) was similar to the past 3 years. Catches of juvenile Slimy Sculpin continue to be low relative to historic catches and the timing of their decline coincides with the proliferation of Round Goby. Additionally, we found a strong negative relationship between trawl catches of Round Goby and near-shore native benthic and demersal fishes such as Trout-perch, Johnny Darter and Spottail Shiner. The introduction of Round Goby and the reappearance of native Deepwater Sculpin have shaped the Lake Ontario benthic prey fish community.