The money is reported to have been given to the campaign group Christians for Independence, which is linked to Yes Scotland.
Scottish Green party leader Patrick Harvie, a high-profile Yes campaigner, called on the money to be handed back, pointing to the businessman's 2000 campaign to retain a law banning the promotion of homosexuality in schools.
Mr Harvie said: "I'd encourage them to ask themselves whether it's appropriate to take money from someone who - albeit some years ago - thought it legitimate to run a privately funded, entirely biased mock referendum to try to influence the outcome of a parliamentary process.
"He showed no respect for legitimate democratic practices," he added.
However, Dave Thompson, the SNP MSP convener of Christians for Independence, defended its donation from Mr Souter.
He said: "Everyone in Scotland who lives here and sees their future here has a right to be heard in this referendum right across the board - all different opinions deserve and must be heard."
Meanwhile, the Yes campaign received a boost when the Rebus actor Ken Stott revealed he backs independence.
Singer Paulo Nutini has also said Unionist "scaremongering" could tip the balance towards a Yes vote - although he declined to say how he planned to vote.