"Do we really want someone with that kind of character?" said Kentucky Sen. Rand Paul. "There's a sophomoric quality that is entertaining about Mr. Trump," Paul added, scolding Trump for going after people's appearance.
Trump responded by doing what he does best: attack.
"I never attacked him on his looks and believe me there's plenty of subject matter right there," Trump said to Paul.
Other candidates quickly jumped in.
"We don't need an apprentice in the White House -- we have one right now," said Wisconsin Gov. Scott Walker.
Former Hewlett-Packard CEO Carly Fiorina, meanwhile, labeled Trump a "wonderful entertainer," before promising that the 2016 campaign would reveal the "judgment and temperament of every single one of us."
And soon enough, the fireworks were flying between Trump and Jeb Bush.
The former Florida governor, who has a lot riding at this CNN debate with his poll numbers stalled, accused Trump of buying influence.
"You got Hillary Clinton to go to your wedding," Bush said.
The exchanges marked a shift in tone for Republican candidates since the last debate in August, when they were more hesitant to take him on directly. By Wednesday, those reservations were gone, a sign of how seriously Trump's competitors are taking his candidacy after he shocked the political world over the summer by dominating the primary battle.
The debate comes as Trump is suddenly facing stiff competition from retired neurosurgeon Ben Carson, who is surging in the polls. Still, the debate is emerging as a fight between Trump, Fiorina, Bush and Paul.
Fiorina, whose breakout performance at last month's debate helped her land a spot in tonight's main debate, is intent on upstaging Trump.
Trump has attacked Fiorina's business record as the CEO of Hewlett-Packard while she has slammed Trump for being light on substance.
Their sparring intensified over the past week after Rolling Stone published an interview with Trump in which he dismissed Fiorina by saying, "Look at that face! Would anyone vote for that?"
Fiorina shot back: "I am proud of every year and every wrinkle."
Bush may have the most at stake. Despite his $100 million bank account and record as a two-term governor running one of the country's largest -- and most complex -- states, Bush is tumbling in the polls.
After a lackluster performance last month, Bush will have to prove that he can turn his troubled campaign around. He'll do that, in part, by taking on Trump directly after spending much of the summer ignoring his presence.
Bush is already taking a more aggressive stance. In recent interviews and posts on social media, the former Florida governor has repeatedly questioned Trump's conservative bonafides, slamming the businessman on immigration, health care and taxes.
At the earlier debate, Louisiana Gov. Bobby Jindal, former New York Gov. George Pataki, South Carolina Sen. Lindsey Graham and former Pennsylvania Sen. Rick Santorum debated thorny political issues like immigration -- an issue Trump has made central part of his campaign rhetoric -- and national security.
When the conversation turned to the controversial issue of "birthright citizenship," Graham said there were certain "rich Asians, rich people from the Mideast" that were "bastardizing citizenship."
Jindal, meanwhile, defended his policy views on immigration, repeatedly asserting that he did not support amnesty.
In another heated exchange, Pataki and Santorum butted heads about a Kentucky county clerk's decision to refuse issuing marriage licenses to same-sex couples.
The woman, Kim Davis, has reignited a national debate about a Supreme Court ruling earlier this year that legalized same-sex marriage across the country.
Santorum called the Supreme Court ruling "unconstitutional," and said there is no more important right than the ability for a citizen to freely exercise his or her conscience.
But Pataki said he would have fired Davis for violating the law.
"I didn't agree with the Supreme Court's decision but it is the law of the land," Pataki said.
While the four candidates traded barbs over numerous issues, there were also calls for the GOP to focus on the ultimate prize of taking back the White House.
"If it is Hillary Clinton, Joe Biden or Bernie Sanders (that becomes president), they're going to pick people we're going to disagree with all the time," Graham said in reference to Supreme Court nominations. "Please understand that we have to win this election."