The man who bellows "Order! Order!" to keep irritable MPs in line has been particularly critical of the government's decision to postpone a historic vote on the deal expected this week, calling the decision "deeply discourteous".
Cabinet ministers have privately expressed their unhappiness at the way he has run Brexit debates, a government source said, and Commons Leader Andrea Leadsom broke cover on Tuesday to imply he was biased against them.
"He's made his views on Brexit on the record, and the problem with that of course is that the chair's impartiality is absolutely essential," said Leadsom, the minister responsible for steering laws through parliament.
Like many parliamentarians, Bercow, who was first elected as a Conservative MP in 1997, supported Britain staying in the European Union in the 2016 EU membership referendum.
Since the government announced the delay on Monday in angry scenes in parliament, Bercow granted opposition MPs an emergency debate on Tuesday to vent their frustration.
"He was always likely to be an interventionist speaker over Brexit, and in this instance, he is making life as uncomfortable for the government as he possibly can," wrote Isabel Hardman, a commentator with The Spectator magazine.
Last week, Bercow also made time for opposition parties to argue that ministers were in contempt of parliament for failing to publish its full legal advice on the Brexit deal.
May's spokesman declined to explicitly endorse him on Tuesday, saying: "Established convention is that the speaker must remain politically impartial at all times. It is for the House to determine if this is not the case."
Bercow assumed the neutral role of speaker nine years ago, and has sought to give backbench MPs more of a say through emergency debates and urgent questions to ministers.
But this has made him a thorn in the government's side and May's predecessor David Cameron tried in vain to get rid of him.
He particularly enraged many Conservative MPs with an extraordinary broadside last year against US President Donald Trump.
He was also criticised earlier this year over a sticker in his car saying "Bollocks to Brexit", which was later reported to have belonged to his wife Sally.
He has clashed with Leadsom over debates, and in May was accused of calling her a "stupid woman" -- something he denied.
Bercow also faced calls to quit for failing -- as the top official in the Commons -- to do enough to tackle bullying and harassment in parliament, but opposition Labour MPs defended him.
His importance will likely increase in coming weeks as MPs use different parliamentary mechanisms to assert more control over the Brexit process.
If and when May brings her deal back to MPs, Bercow will be able to choose which amendments go to a vote.
If the deal fails, opposition MPs may also seek to amend other legislation to force May to alter course, all of which could be in the hands of the speaker.