The Conservatives are reeling after a massive swing saw Owen Paterson's old seat in North Shropshire lost to the Lib Dems, a result Mr Johnson says he takes full responsibility for.
It came amid speculation that Labour had stood aside for the Lib Dems, returning the favour from the recent Old Bexley and Sidcup by-election which saw Sir Ed Davey's party allow Labour a free run.
Shadow Chief Secretary to the Treasury, Pat McFadden, said it was "devastating" for the Conservatives to lose in North Shropshire, where the party held a near-23,000 majority from 2019.
The Wolverhampton South East MP, said: "In every by-election there is a basic question - do the voters want to support the party that holds the seat or send them a message?
"If they want to send a message voters normally work out how to do that by supporting one of the challengers. That's what happened here.
"If you take North Shropshire and the Old Bexley and Sidcup by-election a couple of weeks ago it looks like tactical voting is back, at least to some degree. That could be bad news for the Tories."
Mr McFadden said the Tory vote had been hit by the Prime Minister showing "open contempt for the public health rules he urged the rest of the country to abide by".
In reference to Boris Johnson's botched attempt to back Mr Paterson after he had breached parliamentary standards, he added: "And remember, this by-election only happened because of his attempt to rip up the rules relating to MPs carrying out paid lobbying."
John Spellar, Labour MP for Warley, suggested Labour's performance in North Shropshire – which saw the party poll less than 10 per cent of the vote – was impacted by favourable coverage for the Lib Dems from the London-based media.
He added: "What was completely clear was that the public, for a whole number of reasons, wanted to give the Government – and particularly Boris Johnson – a kicking."
Marco Longhi, Conservative MP for Dudley North, said: "I think what the people of Dudley want to see from me, and what they want to see from Number 10, is a focus on what really matters to them.
"I'm completely focused on making sure that Dudley people get the best possible opportunities in terms of jobs and skills for the future.
"People have come through a really difficult period of uncertainty. This virus was not in the Conservative Party manifesto and I think a lot of people will reflect on that and on what Number 10 has done to combat the virus.
"This is what really matters to people. I'm sure the Prime Minister gets it and we need to make sure we double down and deliver."
The North Shropshire by-election saw Helen Morgan claim victory after receiving nearly 6,000 more votes than Conservative candidate Neil Shastri-Hurst.
Following her win she attacked Mr Johnson, saying his Government was run on "lies and bluster" and that it would be held accountable.
She also thanked Labour supporters for "lending" their votes to the Lib Dems.