Rotherham's Sky Bet Championship match against Middlesbrough on St Stephen's Day has been postponed because of more confirmed Covid-19 cases, the club announced on Wednesday.
The Millers had already seen their home fixture against relegation rivals Derby on Saturday called off after one player tested positive for coronavirus and other squad members showed symptoms.
Rotherham confirmed "a number of further positive cases of Covid-19" within the first-team squad meant the club were also now unable to fulfil the Boxing Day fixture at the Riverside Stadium.
The EFL will now open an investigation into the circumstances surrounding the cancelation of the game, as it does with all such postponements.
A statement on Rotherham's official website read: "Following a number of further positive cases of Covid-19 confirmed last night within the Rotherham United first-team squad, we have reluctantly - but with the safety of our players and staff at the forefront of our minds - informed the EFL and our opponents, Middlesbrough, that we will be unable to fulfil our Boxing Day fixture.
"Paul Warne's side were set to face Boro at The Riverside on Saturday 26th December 2020, but following an increased series of testing throughout the early stages of this week, brought about by the postponement of last weekend's fixture against Derby County, the club have, following consultation with the EFL doctors, been left with no choice but to inform the EFL of our decision to rearrange the game against Middlesbrough.
"The club will continue to monitor the severity at which the virus has spread - alongside the health and well-being of our first-team squad and those connected to them - whilst remaining in contact with our upcoming opponents and the EFL on the matter.
In their statement on the matter, the EFL said that "revised dates for the rearranged fixtures will be confirmed in due course."
Elsewhere, Portsmouth's next two Sky Bet League One fixtures have been postponed due to five confirmed cases of Covid-19.
League leaders Pompey were due to host Swindon on Saturday before a trip Bristol Rovers on December 29.
The club announced that the positive tests affect members of playing and backroom staff, including defender Jack Whatmough, who missed Friday's win at Hull after feeling unwell.
A statement from the English Football League read: "The club informed the EFL that they would be unable to fulfil the fixtures following recent positive Covid-19 tests and the requirement for players and staff to self-isolate as per EFL and Government guidelines in order to mitigate against the risk of further infection.
"Revised dates for the rearranged fixtures will be confirmed in due course."