Harpacticoid copepods can be a substantial component of the meiobenthic community in lakes and serve an ecological role as detritivores. Here we present the first species-level lake-wide quantitative assessment of the harpacticoid assemblage of Lake Ontario with emphasis on the status of nonindigenous species. Additionally, we provide COI-5P sequences of harpacticoid taxa through Barcode of Life Data System (BOLD). Harpacticoids were collected at depths from 0.1 to 184 m and from a range of substrates from August to September 2018 as part of the Cooperative Science and Monitoring Initiative (CSMI) offshore benthic survey. Twenty-six meiobenthic samples were analyzed using microscopy for community composition analysis of harpacticoids. We found thirteen indigenous and three nonindigenous species of harpacticoid, with the introduced species dominating at shallow depths. The community transitioned from nonindigenous to indigenous species dominance as depth increased. Nonindigenous species accounted for 79% of the community (by abundance) at depths <20 m, 55% from 20 to 40 m, and only 24% at depths >40 m. The nonindigenous species encountered included the first detections of Schizopera borutzkyi (Monchenko, 1967) and Heteropsyllus nunni (Coull, 1975) from Lake Ontario. S. borutzkyi was the most abundant harpacticoid species in the lake, approaching a maximum density of 50,000/m² and a lake-wide average density of 7,900/m². Numerically important indigenous species included Bryocamptus nivalis (Willey, 1925), Canthocamptus robertcokeri (Wilson, 1958), Canthocamptus staphylinoides (Pearse, 1905), and Moraria cristata (Chappuis, 1929). The prevalence of nonindigenous harpacticoids in the meiobenthos of Lake Ontario suggests further investigations of Great Lakes meiofauna communities are warranted.