Both candidates have been protected by the Secret Service for months, but Trump's latest take on Clinton's security detail brought swift denunciations, particularly from Clinton allies.
"Tonight, Donald Trump once again alluded to violence against Hillary Clinton," said Elizabeth Shappell, spokeswoman for Correct The Record, a pro-Clinton media watchdog group.
"This is a truly deplorable comment that betrays our nation's most fundamental democratic values," Shappell said in a statement.
Stuart Stevens, a Washington-based political consultant who worked on Republican Mitt Romney's 2012 presidential campaign, tweeted: "The Secret Service should investigate this threat."
Trump made a similar comment about Clinton and her armed protection in May while accepting the endorsement of the National Rifle Association, when he said Clinton would end the U.S. Constitution's Second Amendment, which guarantees the right to bear arms.
"Let's see how they feel walking around without their guns or their bodyguards," he told the gun lobby group, speaking about Clinton and her Secret Service detail.
Trump was criticized by opponents last month when he suggested that gun rights activists could act to stop Clinton from nominating liberal U.S. Supreme Court justices, a comment some interpreted as encouraging a political assassination.