Two accounts tweeted claims about incidents alleged to have happened in 2014 and 2015.
The account of one anonymous accuser has been removed from Twitter, but Justin Bieber responded to it, saying: "There is no truth to this story."
The second account is still online. This account is not anonymous.
A spokesperson for Justin Bieber told Newsbeat they wouldn't comment and had nothing to add to the singer's tweets.
Over the weekend, Riverdale actors also denied accusations of a sexual offence made by other anonymous accounts.
An account from a woman known only as Danielle detailed claims of an assault in Houston, Texas on 9 March 2014.
She says she was encouraged to come forward after seeing unrelated claims about an American actor online.
Danielle claims that after a surprise performance in front of a small crowd at a bar, Justin Bieber invited her and two friends to a Four Seasons hotel in Austin,Texas.
She says he took her to a private room where the sexual assault happened.
She says she did not tell anyone for years, but has now had "multiple therapy sessions" and spoken to close friends and family.
These tweets have been deleted but screengrabs of her comments continue to be shared.
Justin Bieber responded to the allegation "after talking with [his] wife and team", with a denial.
In a series of tweets, the singer posted screenshots and articles from the night the alleged 2014 assault took place, which show him with his then-girlfriend Selena Gomez.
He says they did not stay at the Four Seasons hotel but at an AirBnB with some friends.
He also shared a screenshot of a tweet which claimed he was spotted at the Four Seasons restaurant on 10 March, the day after the alleged assault. That Twitter account appears to have been deleted.
Newsbeat has contacted Four Seasons for comment.