The Conservative leader in the Welsh Parliament Paul Davies has quit after being seen drinking with other politicians in the Senedd, days into a pub alcohol ban.
Tory MS Darren Millar also announced he was stepping down as chief whip.
They were seen drinking in early December, four days after a ban on pubs serving alcohol on their premises.
Mr Davies said he was stepping down "for the sake of my party, my health and my own conscience".
"I simply cannot continue in post," he said, adding he would leave with "immediate effect".
Both will remain as members of the Senedd.
An investigation by the Senedd's authorities found that four members of the Welsh Parliament drank alcohol on its premises during the Wales-wide alcohol ban.
The Senedd has referred the "possible breach" of Covid rules to Cardiff council and its own standards watchdog.
Mr Davies said: "I am truly sorry for my actions on the 8th and 9th December.
"They have damaged the trust and respect that I have built up over 14 years in the Welsh Parliament with my colleagues and the wider Conservative Party but more importantly with the people of Wales."
A third member of the Senedd, Labour's Alun Davies, has also apologised and has been suspended by his party.
BBC Wales has asked for clarification as to the identity of the fourth Senedd member investigators have referred to.
On Friday, when the Conservative group in the Senedd met to discuss the incident, Mr Davies offered his resignation but won the "unanimous" support of group to stay in post.
But it is understood the declaration of support was made before Tory group members had seen the Senedd investigation's conclusion.
Announcing he was stepping down, Mr Davies said during the incident he and his colleagues had maintained social distancing, adding: "There was no drunk or disorderly behaviour."
He said: "We did not have to be escorted out of the building as some reports have suggested.
"What we did was to have some alcohol with a meal we heated up in a microwave, which was a couple of glasses of wine on the Tuesday and a beer on the Wednesday. I broke no actual Covid-19 regulations."
He said since the start of the pandemic he had "followed the Covid-19 regulations to the letter".
"As with everyone across Wales, I have not seen family members or friends, I've not eaten at my favourite restaurants and, like you, we enjoyed a subdued Christmas compared to other years."
He said he was grateful for the support of his colleagues in the Welsh Conservative Party during "this difficult time", especially his wife Julie and Senedd colleagues who he said had " offered empathy, trust and advice".
He said: "Over the last couple of days, I have been speaking with colleagues in the Senedd and the wider Conservative Party.
"Whilst they have confirmed they do not wish to see me step down, I believe that my actions are becoming a distraction from holding this failing Welsh Labour-led Government to account."
Announcing he was standing down as chief whip, Mr Millar, the MS for Clwyd West since 2007, said: "While I am advised that I did not breach coronavirus regulations I am very sorry for my actions, especially given the impact of the tough restrictions that people and businesses are enduring.
"For this reason, and given that Paul Davies has resigned as Welsh Conservative Group Leader in the Senedd, I have decided to step down from my front bench role in the Welsh Parliament."
Chairman of the Welsh Conservative Party Glyn Davies said: "We accept Paul's decision to step down, and would like to take this opportunity to thank him for his service as leader of the Conservative group in the Senedd."
He said the process to find a successor would now get under way.
Had he stayed in post, Paul Davies would have been a wounded leader heading into the Senedd elections in May.
A set of elections which his party has high hopes for, in the wake of their 2019 General Election performance where they made big inroads into Labour strongholds in the north east of Wales.
His successor will want to turn attention back to Labour's handling of the vaccine roll out, where Tory party members felt they had been making headway until this own goal from their senior team in the Senedd came to light.
But Mr Davies' departure could also bring into the open a broader debate within the party about their stance on devolution and whether they should be more overtly sceptical to head off the challenge from abolitionist parties.
What has the reaction been?
Helen Mary Jones, the Plaid Cymru Mid and West MS, said: "Even when the Tory Senedd group was giving its unanimous support to the leader and chief whip, it was obvious to everyone else that their positions were untenable.
"Everyone will be asking what took them so long. No amount of rearranging the deckchairs will make a chaotic and increasingly right-wing Tory party fit to govern Wales."
Secretary of State for Wales, Simon Hart MP, said he had "greatly enjoyed working alongside Paul over the last year following our best general election result in Wales since 1983".
The Conservative MP for Carmarthen West and South Pembrokeshire continued: "He has played a crucial role in helping link the Welsh and UK Government as we fight the Covid-19 pandemic, and has been undeterred in his pursuit to hold the Welsh Labour Government to account since 2018."
The Conservative MS for South Wales Central David Melding tweeted to say he fully supported Mr Davies' decision.
He continued: "There is dignity in accepting responsibility even when actions were unintentional.
"My hope is that Paul will be given the space and support to rebuild his political career. He has so much to offer the WCP [Welsh Conservative Party] and Senedd."