The pair were believed to be in dispute with the club over pay issues. Yesterday it was revealed that Gretna staff and players had not received their wages on time, with club owner Brooks Mileson seriously ill in hospital.
"Gretna Football Club can confirm that David Irons and Derek Collins have resigned with immediate effect. Director of Football Mick Wadsworth, assisted by Iain Scott and Andy Smith, will take charge of first-team affairs until further notice," read the club website.
The crisis at Gretna may yet deepen as chief executive Morton Muir admitted the club could fall into administration.
Speaking to BBC Sport, Muir said: "There's always a danger of going into administration but we've had these kind of rumours before. I don't want to paint that bleak picture and we sincerely hope that, with the amount of effort that's going on between the club and the Mileson family, that administration is not going to be the case."
"You ca n never say never in football but let's say we're encouraged about what's happening at the moment.
"We want to get on with what we've been good at at Gretna Football Club and that's playing football and winning matches."
Muir added that much depends on the continued involvement of owner Mileson.
"The support and financial input that Brooks has given to Gretna has been well documented for many years. The payroll problems have been created by Brooks being ill and getting the release of funds to get it processed.
"That's the main crux of it as Brooks puts the payroll through on a weekly basis. If there's a criticism here then it's that there isn't a system in place to rectify that."