The SNP has seized on remarks by Lord Ashdown, who was critical of the business ethics of companies such as Vitol, over which Better Together's leading donor Ian Taylor presides.
The former Liberal Democrat leader acknowledged that critics were entitled to raise questions on the issue. Now the Nationalists have called on the party's Scottish leader Willie Rennie to declare where he stands on the issue.
Lord Ashdown was UN High Representative in Bosnia when he banned many senior politicians from office over corruption which sheltered war criminals.
Vitol, the world's biggest oil trading firm, has admitted paying $1 million to Serbian warlord Arkan and later struck a deal with a minister in the Bosnian Serb Government just months before Lord Ashdown barred the minister involved from public office for sheltering war criminals.
In both cases the company denied breaking any law and stressed that in the Bosnia deal the contract was struck with ministers of a recognised, legitimate government.
Vitol said there was no way it could have known the actions that would be taken just months later by the UN.
Lord Ashdown said of this: "Anybody who did business in the Balkans who didn't realise there were networks of corruption that extended into any and all governments was naive."
He went on to be even more critical of companies dealing with these regimes.
The peer added of concerns over the donation to Better Together: "Wherever you are coming from, it's a perfectly legitimate line to pursue."
The SNP's Westminster leader, Angus Robertson, said: "The intervention by Paddy Ashdown is very significant, and puts Willie Rennie in a position where he must be upfront and declare if he agrees with Lord Ashdown, or if his loyalties are with the Tory-funded anti-independence No campaign.
"People voting in the referendum in September 2014 are looking for transparency and honesty from the No campaign, and this presents a perfect opportunity for Willie Rennie to regain some much needed credibility for him and his party."
A spokesman for Mr Rennie said: "In his desperation to avoid discussion of his flawed plans to break up the UK, Angus Robertson is distorting Paddy's com-ments. Paddy backed Better Together and did not call for the donation to be returned.
"Willie Rennie supports Ian Taylor's personal donation to Better Together. Ian Taylor has made a significant contribution to Scottish life, charity and Harris Tweed. He disputes the nationalists' claims."
The SNP is determined to keep the issue going at Holyrood. Cathcart MSP James Dornan, who raised the issue in the Chamber last week, has now written to Lord Ashdown.
He writes: "I hope you will agree that it is imperative that incumbent on all sides to ensure that Scotland's referendum is run to the highest of international standards, and that includes how both campaigns are funded."
Mr Dornan also urges Lord Ashdown to use his "influence to persuade colleagues in Better Together to re-think their acceptance of this donation".