Former Tory cabinet minister Lord Tebbit has accused the BBC of becoming a "supporter of a foreign organisation called the European Union".
Lord Tebbit urged ministers to tell the BBC to "get your act in order" because they owed a duty of impartiality over Brexit to viewers and listeners.
However, Digital, Culture, Media and Sport minister Lord Ashton of Hyde said ministers must keep out of editorial decisions by the corporation.
Lord Tebbit suggested there was "something wrong" when out of 4,275 guests talking about the EU on BBC Radio 4's Today programme between 2005-15 only 132 - or 3.2% - were supporters of the UK's withdrawal from the EU.
"Frankly, the BBC has become the supporter of a foreign organisation called the EU," he said to jeers. "Could you just quietly whisper in someone's ear: get your act in order because you do owe a duty of impartiality."
Lord Ashton agreed the BBC did have a duty of impartiality but ministers must keep out of editorial decisions and issues of impartiality were "largely a matter of opinion".
He pointed to a complaint he had read that the BBC had put former Ukip leader Nigel Farage on BBC television's Question Time programme 31 times since it started.
The minister said the BBC was operationally and editorially independent of government but under a duty to deliver impartial and accurate content.
However, Ukip's Lord Pearson of Rannoch said Brexit Secretary David Davis had said recently his job in Brussels was made even more difficult if every time he made an advance it was "undermined by the BBC".
Labour former Lords leader Lord Richard said Lord Pearson and "his ilk" would not be satisfied until every programme stated "down with the tyranist EU".
He said this was "vintage Trump" and amounted to the "last screech of a dying corpse".
Lord Ashton said: "A small portion of Brussels is part of healthy and balanced diet."