The cross-party organisation sent out leaflets to households across Glasgow's Govan area, warning of the impact of independence on shipyards and jobs associated with the UK's defence industries.
It had been distributed two days before Thursday's by-election, triggered by the death of Allison Hunter, a former SNP group leader in the city.
The result saw Labour's John Kane declared the winner in the notorious electoral battleground, beating the SNP's Helen Walker and 12 other candidates.
Labour secured 2055 first-preference votes compared with 1424 for the SNP. Mr Kane, a full-time carer for his disabled partner, was elected at the 13th stage. Turnout was just 20%. But amidst the fall-out, the SNP said Better Together had intervened in a deliberate attempt to undermine the SNP campaign.
Better Together said its mailshot coincided with the publication of the UK's Government's report on defence. A spokesman for the campaign said the letter also went to households in Rosyth, Clydebank and Scotstoun.
But Graeme Hendry, SNP group leader on Glasgow City Council, said: "While we wish Councillor Kane well, it was disappointing to see the Better Together campaign intervened in a council by-election by delivering an eve-of-poll letter to Govan voters.
"This development is concerning for the good running of our democratic processes."
A Better Together spokesman said: "The Nationalists take the term 'sore losers' to a new level. It is utterly desperate."
They denied the mailshots were a one-off. The spokesman added: "Our Patriot system and Blether Together programmes allow us to do exactly that, building on technology used by the Obama campaign."
Scottish Labour deputy leader and local MP Anas Sarwar said: "By ensuring the SNP has no representation in Govan in the council, the people of this area have sent Nicola Sturgeon and the nationalists a strong message - stop obsessing about separation and start putting the interests of the people of Scotland first."
Privately, many in the SNP have accused city council leader Gordon Matheson of insensitivity over his comments following the victory, given the circumstances in which the by-election was held.
Mr Matheson said: "With less than 12 months until the referendum, support for the nationalists has slumped in Glasgow."
An SNP spokesman said: "Hopefully turn-out in future will be above the disappointing 20% in the by-election. The late Allison Hunter will be a hard act to follow and we wish councillor Kane well."