Rebels are discussing whether they can force a vote on the issue under rules that allow MPs retrospective debates about changes to regulations brought in by ministers.
Some of the party's most senior MPs urged Mr Johnson to look again at the rule that forces pubs and restaurants to close early, while a Parliamentary committee demanded to see the scientific evidence on which it was based.
It came amid warnings of further chaos in city centres this weekend when drinkers "pile out of pubs" and flood the streets.
Tory MP Philip Davies clashed with Matt Hancock, the Health Secretary, in the House of Commons as he told him to "start acting like a Conservative".
Mr Davies said: "Is the Secretary of State aware of the damage the arbitrary 10pm curfew is doing to pubs, restaurants, bowling alleys and casinos? Is he aware of the jobs that are being lost, all just to see people congregating on the streets instead and shop staff getting more abuse?
"When will the Secretary of State start acting like a Conservative with a belief in individual responsibility and abandon this arbitrary, nanny state, socialist approach which is serving no purpose at all apart from the further collapse of the economy and eroding our freedoms?"
Another senior party source said: "Under the current rules, Parliament can demand a vote on measures brought in by ministers without Parliamentary scrutiny. We are looking very closely at whether we can force a vote on this, and it's one that the Government might lose."
Steve Baker, the former Brexit minister who helped broker a deal with the Government this week for MPs to be given more say over new restrictions, said: "I'm picking up plenty of dissatisfaction over the 10pm rule among colleagues.
"Many MPs simply don't understand the basis for it. Matt Hancock should look again at whether that rule is working."
Tory MP Greg Clark, the chairman of the Commons science and technology committee, wrote to Mr Hancock demanding that he "detail the evidence and advice informing the decision" to close pubs at 10pm.
Opposition to the policy came from all sides, with the Liberal Democrat MP Daisy Cooper saying: "The evidence is clear that the 10pm pubs curfew has been a hammer blow to hospitality, and turfing crowds of people out of Covid-secure venues onto the streets is putting lives and livelihoods at risk."
Labour's shadow health secretary, Jon Ashworth, asked Mr Hancock what he would do "so we don't see a repeat this weekend of people piling out into city centres, packing out public transport, sometimes all piling in to a supermarket to buy more drink?"
There was also a growing backlash from the pub trade. In a letter to Tory MP Laura Trott, Robert Wicks, of Kent brewer Westerham, said: "For the avoidance of doubt, the Government cannot expect the support of businesses in implementing and monitoring Government policy that is made on the hoof and without scientific backing."
James Calder, the boss of the Society of Independent Brewers, said: "The message the Government is sending to the brewing sector is that despite everything we have done to make our sector safe, despite the fact that pubs and breweries are safe places, despite the fact that the Government's own evidence points towards a tiny minority of transmission coming from hospitality, that we are still the problem.
"We as a sector are being scapegoated because the Government needs to be seen to be doing something, however nonsensical that something is."
Mr Hancock said: "It is perfectly reasonable to make the argument that we should just let the virus rip – I just think that the hundreds of thousands of deaths that would follow is not a price that anyone should pay."