Former minister Tobias Ellwood and backbencher Julian Sturdy described partygate as a “distraction” during a challenging time for Britons.
Mr Sturdy, Conservative MP for York Outer, said the Sue Gray report showed Mr Johnson “has presided over a widespread culture of disregard for the coronavirus regulations”.
In a statement posted on Twitter, he said: “Questions are now being raised about whether the Prime Minister misled Parliament when asked about these events.
“Talking to constituents, it is clear discussions about parties in Downing Street remain a damaging distraction at a time when our country faces massive challenges with war returning to Europe, a global cost of living crisis, and our recovery from the pandemic being more important than ever.
“This is clearly a time when we cannot have any doubt about the honesty, integrity, and personal character of the Prime Minister.
“While I thought it important to wait for the conclusion of the Metropolitan Police investigation and the publication of the Sue Gray report, I am now unable to give the Prime Minister the benefit of the doubt and feel it is now in the public interest for him to resign.”
Conservative former ministerial aide Angela Richardson said the scandal has eroded public trust in politicians and “reflects badly on us all”.
“I am clear that had this been a report about my leadership, I would resign,” the Guildford MP wrote online.
Mr Ellwood was heckled by fellow Tory MPs in the Commons chamber as he questioned whether they could continue to defend Mr Johnson’s behaviour.
A question I humbly put to my colleagues is ‘are you willing day in and day out to defend this behaviour publicly?’
The MP said of the report into lockdown-busting parties in No 10: “This is a damning report about the absence of leadership, focus and discipline in No 10 – the one place where you expect to find those attributes in abundance.
“I’ve made my point and my position very clear to the Prime Minister: he does not have my support.
“But a question I humbly put to my colleagues is ‘are you willing day in and day out to defend this behaviour publicly?’
“Can we continue to govern without distraction given the erosion of the trust with the British people?
“And can we win the general election on this current trajectory? I’m being heckled by my own people.
“If we cannot work out what we’re going to do, then the broad church of the Conservative Party will lose the next general election.
“But my question to the Prime Minister is very clear, on the question of leadership: can he think of any other prime minister who’d have allowed such a culture of indiscipline to take place under their watch and if it did would they not have resigned?”
Mr Johnson’s immediate fate is in the hands of Conservative MPs, and whether enough of them chose to write letters to trigger a vote of no confidence.
Culture Secretary Nadine Dorries told TalkTV’s The News Desk that Mr Johnson would “absolutely” win such a challenge.
Back in the Commons, Mr Johnson focused his answer on his prospects of winning the next election.
He said: “I think the answer is overwhelmingly and emphatically yes, we are going to go on and win the next general election because we’re going to get on with the job.”
SNP Westminster leader Ian Blackford, who accused Mr Johnson of adopting a “sinister pattern of evasion”, appealed to Tory MPs to oust the PM.
He said: “I hope they will bear in mind the now infamous Government advert featuring a desperately ill Covid patient, it said: look into her eyes and tell her you never bend the rules.
“If they don’t submit a letter, if they don’t remove this Prime Minister, how will they ever, ever look at their constituents in the eye ever again?”
Plaid Cymru Westminster leader Liz Saville Roberts said: “In this farce of a parliamentary system it’s now all down to Tory MPs… for them to grow a backbone and oust this moral vacuum of a Prime Minister.
“Will he spare them the trouble and resign?”
Mr Johnson replied: “No.”
Conservative MP John Baron (Basildon and Billericay) asked Mr Johnson if he believed his statements on “partygate” in the Commons had “passed the test of reasonableness”.
He said: “I believe both leaders have a lot to answer for with regards to this issue. The British army teaches you, or certainly believes at its very core, that you serve to lead and you lead by example.
“Given the extent of rule-breaking in Number 10, does he believe that what he has said to the House since about their being no rule-breaking passed the test of reasonableness?”
Mr Johnson restated his argument that he believed he was attending work events.
Conservative former cabinet minister Robert Jenrick (Newark) said it was “now time to turn a page” and spoke of the actions of “brilliant” civil servants to create the shielding programme in the early stages of the pandemic.
He said: “These achievements and others should mean that nothing in this report is a stain upon the character of the thousands if not hundreds of thousands of civil servants whether in Number 10, other government departments or across the country who helped steer this country through the pandemic.
“Secondly difficult though this is to say for many, with a war in Europe, with an economic crisis, with the challenges this country faces, is it not really true that it is now time to turn a page and for this country, our politics and this House to move forwards.”
SNP Pete Wishart (Perth and North Perthshire) said there had been “no attempt at remorse” from Mr Johnson, adding: “Surely if he was half the man that he thinks he would summon that self-respect and just go.”
Labour MP Afzal Khan (Manchester Gorton) said his mother, father-in-law and mother-in-law died from Covid-19, adding: “The laws broken by the Prime Minister, the Chancellor and others were not victimless crimes.”