The married player, who is referred to as CTB in court documents, is said to have had a "sexual relationship" with Imogen Thomas, a former contestant on the Big Brother reality TV show.
He obtained an order preventing The Sun newspaper from revealing his name last month and last week launched proceedings against Twitter and "persons unknown" after users claimed to have identified a number of individuals said to have taken out gagging orders to protect their identities.
The legal bid comes after a Twitter user identified a number of people said to have taken out gagging orders, fuelling the privacy debate and highlighting the difficulty of enforcing injunctions. An estimated two million people are believed to have seen the list.
Speculation surrounding the footballer's identity reached new heights yesterday, with one player's name being mentioned on Twitter at a rate of up to 16 times a minute. One popular message being re-tweeted on the micro-blogging site, which made explicit mention of him, stated: "X X is suing Twitter. I can't Imogen why."
Scores of users were also claiming to identify a woman alleged to have had an affair with former Royal Bank of Scotland chief executive Sir Fred Goodwin.
Social media experts said the trend demonstrated how the use of super-injunctions to hide alleged affairs have become "pointless".
Social media expert Olly Mann told Scotland on Sunday: "The use of sites like Twitter makes super-injunction orders pointless to a generation of people, and it seems a very tech-savvy, media-savvy group think a secret can't be kept from them."
However, the chief executive of the major law firm representing the footballer said that the action being taken against the micro-blogging site was akin to asking Royal Mail to disclose the identity of someone sending "unpleasant" packages.
Rod Christie-Miller, who is a partner at Schillings, said that Britain was now at an "important juncture" in terms of how it deals with privacy laws.
Christie-Miller, who is not personally representing the footballer, said: "It's not really very much different from asking the Post Office to disclose who's been lobbing unpleasant things through a PO box.
"It's important, it's interesting I think, because it concerns social media."
Schillings said the footballer was not suing Twitter, and issued a statement to clarify the reason for taking the action.
"An application has been made to obtain limited information concerning the unlawful use of Twitter by a small number of individuals who may have breached a court order," it said.
Christie-Miller, who sat on a committee headed by Lord Neuberger to review the use of super-injunctions, said the courts and parliament had to work out how to balance privacy with freedom of expression.
He said: "There are plenty of people who find themselves in the public eye through no fault of their own. We've got to think about people who have been arrested but ultimately not charged with criminal offences. A few very high-profile cases like that have been wrongly targeted by the media.
"We have a media culture at the moment which is very invasive, a tabloid culture.
"It's right that many things are published but there has to be a balance between publishing rights and privacy rights."
A spokesman for Twitter, which is based in San Francisco, said: "We are unable to comment."
Last year Twitter users angered by the conviction of a man who jokingly threatened to blow up an airport showed support by republishing the words that landed him in trouble.
Paul Chambers, an accountant, lost an appeal against his conviction and 1,000 fine for a comment he made in jest when he was concerned he might miss a flight to Belfast.
Under the hashtag #IAmSpartacus - a reference to the film in which Spartacus's fellow gladiators show their solidarity with the protagonist - thousands of people copied the original message.
Meanwhile, the Financial Services Authority has announced it is to launch a new investigation into the failure of RBS in light of the revelations about Goodwin's private life.
Details of his alleged affair were revealed with the partial lifting on Thursday of a super-injunction granted to Goodwin. It allowed the media to disclose that the 52-year-old father-of-two had tried to hide a "sexual relationship" but not to name his mistress.