The two rivals are vying to emerge as the chief alternative to the absent former president Donald Trump, who has maintained a commanding lead in opinion polls ahead of Iowa's January 15 contest.
"She caves anytime the left comes after her, anytime the media comes after her," DeSantis said of Haley on Wednesday night during the first answer of the evening, as he sought to explain why voters should back him despite Trump's dominant position.
DeSantis boasted about legislation he has passed in Florida banning gender-affirming medical care for transgender youth and accused Haley of opposing the law, an assertion Haley denied.
"He continues to lie about my record," she said.
Former tech entrepreneur Vivek Ramaswamy and former New Jersey Governor Chris Christie also took the debate stage in Tuscaloosa, Alabama. But all eyes were on DeSantis and Haley as the narrowing Republican field runs short on time to derail Trump's march to the 2024 nomination.
As with the first three debates, the former president - leading by more than 40 percentage points in most opinion polls - skipped Wednesday's event, instead attending a fundraiser in his home state of Florida.
Trump's absence deprived his rivals of an opportunity to confront him face to face and again sent the message that he deems his challengers unworthy of his attention.
DeSantis holds a small advantage over Haley in national polls. But Haley has been closing the gap, and has a substantial edge over DeSantis in New Hampshire and her home state of South Carolina - crucial states in the nomination battle because they're among the first to pick a nominee.
The two are effectively tied in Iowa.
With no additional debates currently scheduled, Wednesday's televised clash could be the last chance for Haley or DeSantis to land a lasting blow against their opponent in front of a national audience.
Ramaswamy, who has aligned himself closely with Trump, teamed up with DeSantis to go after Haley, attacking her as "corrupt" for making money on speeches and serving on the board of Boeing.
"I love all the attention, fellas," she said, before defending her work with Boeing and suggesting her rivals were jealous she had the backing of major donors.
Only Christie appeared willing to attack Trump directly, criticising him for saying during a Fox News town hall on Tuesday that he would not become a dictator except on "day one".
"I've got these three guys who are all seemingly to compete with, you know, Voldemort, he who shall not be named," Christie said, referring to the unspeakable villain in the Harry Potter stories.
"They don't want to talk about it."
He also came to Haley's defence by going after Ramaswamy for arguing that the United States should not support Ukraine in its war against Russia, saying the entrepreneur was coming across to viewers as "the most obnoxious blowhard in America".
