The group, which includes the former CIA director Michael Hayden, said Mr Trump “lacks the character, values and experience” to be president.
Many of the signatories had declined to sign a similar note in March.
In response, Mr Trump said they were part of a “failed Washington elite” looking to hold on to power.
The open letter comes after a number of high-profile Republicans stepped forward to disown the property tycoon.
Mr Trump has broken with years of Republican foreign policy on a number of occasions.
The Republican candidate has questioned whether the US should honour its commitments to Nato, endorsed the use of torture and suggested that South Korea and Japan should arm themselves with nuclear weapons.
“He weakens US moral authority as the leader of the free world,” the letter read.