The Cord coalition has issued a three-day ultimatum for Health Cabinet Secretary Cleophas Mailu and his Principal Secretary Nicholas Muraguri to vacate office.
Cord, on Friday, said the two have until Monday to vacate office or be forced to resign.
At press conference at Orange House in Nairobi, Ugunja MP Opiyo Wandayi and Homa Bay Women Rep Gladys Wanga said the level at which corruption is being condoned by the Jubilee administration had reached peak.
"We wish to put on notice the current Cabinet Secretary responsible for Health Mr Cleophas Mailu and his Principal Secretary Mr (Nicholas) Muraguri," Wandayi said.
"What has come out is evidence enough that the Kenyan public has once again lost colossal sums of money running into billions of shillings under their watch. The internal audit report has documented all this and therefore our position is that we don't need anymore shenanigans, we don't need anymore audits and we don't need anymore investigations."
"The facts have been laid bare. What we expect the law enforcement agencies is to move swiftly to Afya House and arrest all the culprits who are responsible for the theft of the Sh5.3 billion. They have the entire weekend to clear their desks, pack their belongings and vacate Afya House. Failure to do so, come Tuesday November 8, we will call Kenyans to the streets of Nairobi. The mass action will continue until the two vacate office."
On Wednesday, attempts by civil society groups to stage a demonstration to force the two leave office failed as police dispersed protesters.
Several people were arrested and detained.