The allegation will pile pressure on Johnson over what he knew when he appointed Christopher Pincher to a role involved in enforcing discipline and offering pastoral care in the governing Conservative Party.
Many of his lawmakers are increasingly frustrated at defending what some say is a scandal-ridden administration and could renew attempts to try to unseat the prime minister just a month after Johnson survived a confidence vote.
The main opposition Labour Party accused Johnson of "dragging British democracy through the muck".
"No 10 keep changing their story and are still not telling the truth," Simon McDonald, who served as permanent under-secretary to the Foreign Office at the time Pincher was a junior minister there, said in a tweet with a letter to the Parliamentary Commissioner for Standards he posted on the site.
McDonald, a British diplomat for decades and now a member of the upper house of parliament who would rarely speak out on internal government matters, said there was an investigation into Pincher in 2019 and that "Mr Johnson was briefed in person about the initiation and outcome of the investigation".
Pincher resigned as deputy chief whip last week saying he had drunk too much, embarrassed himself and "caused upset" to people. British media reported that Pincher had sexually assaulted two male guests at a London club.
Newspapers have since reported he had faced several previous allegations of sexual misconduct. His office said Pincher, who has been suspended by the Conservative Party, was now on a leave of absence focusing on receiving medical support and could not be reached.
Last week, Johnson's office said the prime minister was not aware of any specific complaints against Pincher before appointing him. On Monday, his spokesman said he was aware of "some allegations that were either resolved or did not progress to a formal complaint".
"In the absence of a formal complaint it was not appropriate to stop an appointment on the basis of unsubstantiated allegations," he said on Monday.
McDonald, however, said Johnson's office was making "inaccurate claims".
"In the summer of 2019, shortly after he was appointed minister of state at the Foreign Office, a group of officials complained to me about Mr Pincher's behaviour ... An investigation upheld the complaint; Mr Pincher apologised and promised not to repeat the inappropriate behaviour," he said in the letter.
"Mr Pincher was not exonerated. To characterise the allegations as 'unsubstantiated' is therefore wrong."
Labour's deputy leader, Angela Rayner, said Johnson's "desperate attempts to cover up what he knew about sexual assault complaints against Chris Pincher before appointing him have been blown out the water".
"Boris Johnson is dragging British democracy through the muck. His appalling judgment has made Westminster a less safe place to work," she said in a statement.
