Within hours of the body signing up as a No supporter, Tony Banks, the chairman of the Balhousie Care Group and leader of the pro-independence group Business for Scotland, announced his company would leave.
Martin McAdam, chief executive of wave energy company Aquamarine Power, also said that, as a consequence of the CBI's actions, "we can no longer remain members".
The electrical contractors union Select, which has almost 15,000 members, urged the CBI to change its mind or face further action.
But the business body, which has long been critical of what it says are unanswered questions over independence, said it would not be swayed.
A spokesman said: "While any member deciding to leave is a cause for regret, the CBI is confident we have a mandate from the vast majority of our membership on the question of Scottish independence."
A Better Together spokesman also accused nationalists of attacking "the messenger rather than debate the issues".
The row erupted after the organisation registered with the Electoral Commission, as backing a No vote in September's referendum.
It is a requirement for organisations planning to spend more than £10,000 on campaigning and also gives the CBI access to the voters roll and the right for its representatives to attend polling stations and vote counts.
Business for Scotland said it had succeeded in its drive to force the CBI to formally sign up as a No campaigner.
Mr Banks added that the registration had now put the CBI "in an untenable position" and was a "major blow" for independence opponents.
He added: "(The CBI) has attempted to hijack its members without proper consultation on their views and many must now feel they are in an impossible position."
Business for Scotland said that it too now intended to also register, as a supporter of the Yes campaign.
The organisation's chief executive Gordon MacIntyre-Kemp said: "This is a major victory for Business for Scotland.
"We have been calling in public and requesting in private that the Electoral Commission (Scotland) ask the CBI to register as a No campaigner.
"That they did so on Thursday is a major victory for us and a blow to the No campaign."
A CBI spokesman said: "The CBI has clearly stated its position in the Scottish referendum debate."