It followed a similar letter released in March, but the wording of the latest condemnation was both broader and stronger, and the list of names longer.
The alarm among the national security establishment followed Trump's comments about encouraging Russia to hack Clinton's emails, and his refusal to embrace the principles of Nato.
Trump responded with a statement deriding the signatories as members of "the failed Washington elite" who "deserve the blame for making the world such a dangerous place".
"These insiders - along with Hillary Clinton - are the owners of the disastrous decisions to invade Iraq, allow Americans to die in Benghazi, and they are the ones who allowed the rise of Isis," he continued.
It came as Trump faced a new threat from within his own party.
Evan McMullin, 40, a Republican official and former CIA counter-terrorism officer, announced he would run as an independent candidate for president. He could potentially take away a significant number of votes from the billionaire in some states.
McMullin launched his campaign with the slogan "It's never too late to do the right thing".
He has been a member of the "Never Trump" movement within the party and reportedly had the backing of key Republican donors.
McMullin condemned Trump for his authoritarianism and anti-Muslim rhetoric, which he said would make the US "weaker".
He added: "America deserves much better than either Donald Trump or Hillary Clinton."
McMullin spent 11 years in the CIA but has never held elected office. Since 2013 he worked as a policy director for the Republican Party in Congress.
- additional reporting Reuters