Lake Ontario boasts a diverse fish community comprised of native and introduced species that support vibrant recreational, commercial and Indigenous fisheries. The effective delivery of a program to assess and control the sea lamprey (Petromyzon marinus) is crucial to achievement of Lake Ontario Fish Community Objectives of rehabilitating native fish stocks while protecting and maintaining the abundance of introduced salmonines. During 2000–2019, the Great Lakes Fishery Commission (GLFC) and its control agents, Fisheries and Oceans Canada (DFO) and the U. S. Fish and Wildlife Service (USFWS), delivered a consistent program of sea lamprey assessment and control. Beginning in 2004, rising sea lamprey abundance and marking rates on lake trout (Salvelinus namaycush) in Lake Ontario coincided with a decline of large lake trout in gillnet surveys. Efforts were undertaken to identify and control important sources of juvenile sea lampreys, including larvae that survived treatment, inhabited deepwater areas, or colonised previously uninhabited stream reaches and tributaries. A renewed reliance on proven conventional controls, including lampricide treatments and barriers, has resulted in consistent suppression of sea lamprey abundance and lake trout marking to prescribed targets in Lake Ontario during 2014–2019. This achievement is unprecedented in the 49-year history of Sea Lamprey Control Program delivery in Lake Ontario, and is attributable to the collaborative efforts of the GLFC, the control agents, and federal, provincial, state, and Indigenous partners.