Oil trader Ian Taylor, who heads up the controversial firm Vitol, gave half a million pounds to the pro-Union group, which faced calls to hand the money back because of concerns about the company's activities after details of the donation emerged last year.
Now a newspaper investigation has claimed Vitol, which has faced criticism over its links to Serbia, Iran, Iraq and Libya, is one of the UK's biggest tax avoiders.
Better Together said the money given to their campaign was a personal donation from Mr Taylor, not from the company, and therefore there was not an issue.
Vitol, meanwhile, said all political donations were an individual matter for Mr Taylor.
But the SNP renewed its calls for the £500,000 donation to be returned to the businessman, with MP Angus Robertson saying it was "unbelievable that the No campaign is happy to accept these funds". In addition to the funding for Better Together, Mr Taylor has also given £550,000 to the Conservative Party.
The Independent newspaper claimed Vitol had used mechanisms to route staff employment through Switzerland to avoid billions of pounds of tax.
The newspaper claimed taped conversations revealed company traders were renamed as third-party brokers to avoid tax, resulting in Vitol paying only around 10 per cent on its £9 billion profits over the last decade.
In a statement, the company said: "Vitol is a global company with 38 offices worldwide. It has an open and transparent relationship with the tax authorities in all the jurisdictions in which it operates.
"These authorities are fully aware of how the business operates. Vitol pays the correct level of tax in accordance with the appropriate legislation in each of these jurisdictions."
Mr Taylor and Alistair Darling, head of Better Together, met on Lewis in autumn 2012 and Mr Taylor later decided to hand over the money - by far the biggest donation received by the No campaign.
Better Together insists that this donation - and the one to the Conservative party - was personal rather than corporate.
The pro-independence arts community website National Collective has documented the controversial background of Vitol, with dealings in Libya, Iran, Iraq and the former Yugoslavia, and reports payments were made to warlord Arkan as an intermediary.
Responding to the allegations of tax avoidance, Mr Robertson, SNP leader at Westminster, said: "It just goes to show that while the UK Government punishes vulnerable and disabled people with cruel benefit cuts, Westminster works for the likes of Ian Taylor.
"No wonder he wants to keep the system exactly as it is, by giving half a million pounds to the No campaign.
"The Westminster system works for the few, for people like Ian Taylor. We want an independent Scotland because it will work for the many."
He added: "We know that Vitol gave the notorious Serb warlord Arkan $1 million.
"Arkan was indicted by the International Criminal Tribunal for the former Yugoslavia for 'wilfully causing great suffering, cruel treatment, murder, wilful killing, rape and other inhumane acts'. This latest charge of tax-avoidance is just the latest episode in the Donorgate story.
"It is unbelievable that the No campaign is happy to accept these funds, and in light of these latest revelations Alistair Darling should at last do the decent thing and hand this donation back.
"The Taylor money was reportedly personally secured by Mr Darling, and if he does not hand this half-million pounds back, he is - as a former Chancellor - effectively condoning tax avoidance."