Macclesfield's game against Crewe on Saturday has been suspended after Town informed the EFL they would be unable to fulfil the fixture.
The announcement had been anticipated after Macclesfield's players released a statement saying they would refuse to play the League Two match due to non-payment of wages.
An EFL statement on Friday afternoon read: "Macclesfield Town has informed the EFL that they will not be in a position to fulfil Saturday's League Two fixture against Crewe Alexandra.
"As a result of this formal notification, the league has been forced to suspend the game and under EFL Regulations, the club is now deemed to be guilty of misconduct and will be referred to an independent disciplinary commission.
"The EFL will now consider the matter of determining when the fixture will be played."
Macclesfield's senior players went on strike for November's FA Cup first-round tie against Kingstonian, which the non-league side won 4-0 against a makeshift Macc line-up.
They returned for the league match with Mansfield having been paid but with problems arising once more as the club face a High Court winding-up petition – backed by former manager Sol Campbell – the Crewe match will not go ahead with the players citing issues of "mental well-being".
A statement issued by the squad on Thursday read: "As a group, we'd like to inform the EFL and Crewe Alexandra Football Club that the game on Saturday won't be taking place despite growing reports that is is 'business as usual'.
"This is down to the fact that salaries have not been paid for last month. We have not trained as a squad since last Thursday due to the fact we have no training ground but, most importantly, the game can't go ahead for the emotional and mental well-being of the players which is currently rock bottom."
The EFL's statement on Friday continued: "The EFL has considerable sympathy with the difficulties faced by all those affected at the club and is continuing to offer appropriate support to players via the PFA and to staff through our partnership with mental health charity Mind.
"In addition to the existing and potential future regulatory action being taken, the EFL will continue to work on assisting in attempting to secure an appropriate resolution to the overall challenge facing the club.
"It has been utilising central payments to ensure players and staff were paid whilst the owner has looked to find a sustainable longer-term solution. It does remain the obligation of the club to find a way to deliver on the commitments it has made to its players, staff and other stakeholders."
The winding-up petition against the club has been adjourned until December 18.
Campbell's barrister Philip Currie told a court hearing his client is owed £182,000 by the club while barrister Raj Arumugam, representing HM Revenue & Customs, said the club owed a "very large" amount in tax.
Macclesfield have become the latest EFL club to experience financial difficulties, with Bury being expelled from the league in August and Bolton only surviving thanks to a last-ditch takeover.
