Incumbent Republican Sen. Marco Rubio and Democratic Congressman Patrick Murphy faced off at the University of Central Florida.
The presidential race between Hillary Clinton and Donald Trump overshadowed the debate in its opening minutes.
Murphy defended the former Secretary of State when asked Monday why Floridians should trust him when they don't trust Clinton, who he is backing.
Murphy says he trusts Clinton with the nation's nuclear code and that she is as qualified as any candidate.
He called Trump the most unfit candidate to run for the White House.
Rubio said he doesn't trust either candidate, but he's asking voters to send him back to Washington so he can stand up to the president, whether Trump or Clinton.
"I know that no matter who wins this election you are going to need people in the United States Senate willing to stand up to the next president of the United States when they are wrong on policy or behavior. I am the only one that will. I have repeatedly done so when it comes to the nominee of my party. Congressman Murphy has not once," said Rubio.
Rubio has said he won't appear with Trump but is still backing the GOP nominee.
Courtesy of our news partner at NBC Miami
Murphy fired back, "No this isn't just a binary choice and there are people that have the courage to stand up to do what's best. And, I appreciate your question because you do have to look your children in the eye 10-15 year from now and explain this position".
The Democratic candidate also criticized Rubio's voting record during his time in the Senate and the Republican's future political aspirations.
"Will you join me tonight and sign a six-year pledge that you are committed to serve this job and won't seek higher office?", Murphy said.
Rubio responded, "I'm going to serve sic years in the United States Senate God willing and I'm looking forward to it."
The focus then turned to Murphy, his qualifications along with his political and professional experience.
"You didn't run saying you were a CPA. You ran saying you are experienced working as a CPA. You've never had a license to work as a CPA in Florida. So, you either you never worked as a CPA or you were working illegally as an unlicensed CPA," questioned Rubio.
The race for Florida's U.S. Senate seat is one of many competitive races that could determine which party controls the upper House after the November 8th election.
Republicans currently have control of the Senate.
Rubio and Murphy won their primary elections by large margins.
An October NBC News/Wall Street Journal Poll shows the Republican ahead of his Democratic challenger by two points.
"Patrick Murphy hasn't been on the big stage, he hasn't been under the bright lights, and this is really going to be a bigger test for him than it is for Marco Rubio," said debate panelist and Politico Florida reporter Marco Caputo.
Five other contenders qualified as candidates for the Florida Senate race.
Non-partisan candidate Steven Machat appeared before a judge Monday after filing a motion asking for an order to allow all non-partisan candidates to participate in Monday's debate.
The judge denied the motion.