Speaking during First Minister's Questions on Thursday, Nicola Sturgeon rejected the idea of using the nuclear deterrent, saying it had the potential to kill "millions or even tens of millions" of people.
In response to a question from SNP MSP Jenny Gilruth, which mentioned Ms Swinson by name, the First Minister was unequivocal in her response.
Ms Gilruth described the answer given by Ms Swinson following the ITV leaders' debate - that she would use nuclear weapons - as "disgraceful".
The Liberal Democrat leader has been criticised for her response, including by the First Minister.
Ms Sturgeon said: "No I wouldn't. I oppose nuclear weapons.
"Whenever that question is asked, it should be pointed out that anybody who used nuclear weapons would be doing something that would potentially lead to the deaths of millions, perhaps tens of millions of people.
"I think nuclear weapons are immoral, I think they're ineffective and I think they're a waste of money.
"I would not countenance their use and I look forward to the day where not just Scotland is free of nuclear weapons, but the world is free of nuclear weapons."
The First Minister had previously tweeted her disapproval at Ms Swinson's position, describing the exchange as "sickening".
During a round of interviews with leaders not included in the main ITV debate on Tuesday, Ms Swinson was asked a series of "quick-fire" questions, including about whether she would use a nuclear weapon or not.
She decisively answered: "Yes."
Ms Sturgeon and her party have repeatedly voiced their opposition to nuclear weapons, including pledging in the 2014 White Paper to remove the Trident deterrent system from Scotland in the first Holyrood term following an independence vote.