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.
However, with problems arising once more as the club face a High Court winding-up petition the Crewe match will not go ahead with the players citing issues of "mental wellbeing".
The EFL statement continued: "The EFL had met members of playing staff at Macclesfield Town alongside representatives of the PFA earlier on Friday.
"During that constructive meeting, it was made clear that the health and wellbeing of both the players and staff had been adversely affected by the ongoing late payment of wages and, as a result, would be unable to focus on on-the-pitch matters.
"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."