Wynne made the declaration after she helped officially open the Louise Temerty Breast Cancer Centre at the Sunnybrook Health Sciences Centre in Toronto.
There has been no announcement on who will carry out the investigation.
Watered-down chemotherapy drugs were given to 990 cancer patients — some for as long as a year.
Patients affected were treated or are being treated at London Health Sciences Centre, Windsor Regional Hospital, Lakeridge Health in Oshawa and Peterborough Regional Health Centre.
"It’s unacceptable that this should have happened; that the doses would not have been accurate," Wynne said. "The minister is pulling together all the people necessary to get to the bottom of it."
Matthews said Health Canada, the Ontario College of Pharmacists, hospital leaders and Cancer Care Ontario are being consulted.
"Every one of those groups will bring a different perspective," Matthews said.
She wants to know whether the problem "is a system issue or a one-incident issue."
Patients were given lower than intended doses of cyclophosphamide and gemcitabine. The chemotherapy is part of a regime for breast and lung cancer as well as lymphoma and leukemia.
The premixed bags contained too much saline solution, which diluted the chemotherapy agent.
Doctors have been reassuring patients that any health consequences are unlikely.
"We must look specifically at what happened," Matthews said. "We owe it to cancer patients to provide the best possible care. We need patients to have confidence they’re getting the best possible care in the world."
On Wednesday, the law firms Sutts, Strosberg LLP and Siskinds LLP announced a class-action lawsuit to promote patient safety and monitoring of chemotherapy delivery.
The Canadian Breast Cancer Network also said it is vital for patients to be informed of the impact of diluted medications on their treatment outcomes and that adequate measures are taken to ensure errors don't occur again.
Matthews and Wynne both said the most important issue right now is the safety and care of patients.
"Each patient is unique and needs to be in touch with their oncologist," the premier said.
Matthews said oncologists are working evenings and weekends to meet with their patients. She said "hospitals are doing an excellent job reaching out to these patients."