Heriot-Watt University and the University of the Highlands and Islands have resigned from the business organisation, while Dundee University has suspended its membership.
The CBI officially registered as a supporter of the pro-union Better Together campaign on Friday, prompting a walkout by universities, public bodies and businesses.
A Heriot-Watt spokeswoman said: "Heriot-Watt University has a strictly neutral position on independence. As such the court of Heriot-Watt has decided that the university should withdraw from membership of CBI Scotland in light of that organisation's decision to register with the Electoral Commission as a supporter of the pro-union campaign in the independence debate".
A University of the Highlands and Islands spokeswoman said: "The University of the Highlands and Islands advised CBI in April 2013 that the university wished to terminate its membership."
A Dundee University spokesman saidwhile it hosted debate from all sides in the discussion around independence, it maintains a strictly neutral position on the issue as an institution.
Yesterday's action leaves Aberdeen's Robert Gordon University as the only higher education institution in CBI Scotland. It has expressed its disapproval at the CBI's decision but has decided to remain a member. Glasgow, Aberdeen, Edinburgh, Strathclyde and Caledonian all resigned in recent days.