The Charity Commission wants to "establish the facts" relating to the Professional Footballers' Association charity as the scrutiny intensifies on chief executive Gordon Taylor.
The non-ministerial government department which regulates registered charities in England and Wales says it is "aware of concerns" regarding the PFA charity which helps support current and former professionals.
Over 200 current or former members of the PFA have backed chairman Ben Purkiss' call for a complete overhaul of the organisation.
It was announced on Wednesday that an independent QC is to conduct a "full and open review" of the union, which Taylor has run since 1981 and is by far the highest-paid union official in Britain.
The union's management said it would work closely with its trustees and management committee, which is made up of 18 player representatives, to finalise the scope and timeline of the review.
The latest PFA charity accounts show staff costs of nearly £3.8million for charity activities but elsewhere it is highlighted: "The charity does not have any employees and therefore no salaries or wages have been paid during the year."
The Charity Commission said in a statement: "The public rightly expect charitable funds to go to causes they are set up to support.
"As regulator we expect all charities to carefully steward funds in the best interests of their charity in order to maximise their benefit to society.
"We are aware of concerns regarding the expenditure of the Professional Footballers' Association Charity and we will be engaging with the trustees to establish the facts.
"Trustees should be able to demonstrate that all decisions around expenditure have been carefully considered in line with the best interests of their charity and those it is set up to help."
Press Association Sport is seeking a response to the Charity Commission's statement.
High-profile former players including Chris Sutton and Robbie Savage have backed the movement for change.
However, others such as Jon Walters, who currently plays as a striker for Ipswich and serves on the PFA's management committee, have launched a staunch defence of Taylor, accusing his detractors of succumbing to "mob rule".
Taylor – paid £2.3million last year – has himself hit back by calling into question Purkiss' right to membership of the union. Purkiss is currently with Walsall but on non-contract terms.
Among the issues Taylor's critics have raised is what they see as a lack of support for players in financial difficulties or those struggling with mental health issues.
Another line of attack has been the PFA's slow response to football's dementia crisis, with campaigners such as Jeff Astle's daughter Dawn telling Press Association Sport that she "begs" players to replace Taylor and focus the union's significant resources on areas such as research and support.