It said they would not win work with TfL unless they could prove that they would not be distracted from delivering improvements to the Underground.
"Will they continue to find it hard to win contracts with us? Yes they will," said TfL commissioner Peter Hendy.
Atkins, Balfour Beatty and Bombardier Transportation along with EDF Energy and RWE Thames Water are Metronet's shareholders. They also carry out the bulk of Metronet's much criticised upgrade work on the Tube.
Hendy has been an outspoken critic of Metronet. In June he said that he was prepared to publicly blame its shareholders for their failure to perform on Tube projects.
His comments this week came after Balfour Beatty secured the long-awaited main works contract for the £1bn East London Line extension in joint venture with Carillion.
Hendy said that the contract award was delayed because TfL had sought extra reassurances of Balfour Beatty's capabilities.
"We wanted to make damn sure that giving them [Balfour Beatty] another massive contract wouldn't mean they'd f**k up the massive contract they already have any more than they already are doing."
Balfour Beatty said it had won the East London Line contract on merit.
"We had regular contact with Peter Hendy involving proper and appopriate dialogue. We tendered for the East London Line contract in our usual professional and thorough manner and we believe we won the contract on merit," it said.