NBC said the spot infringed on its copyrighted content and that it did not give Obama's campaign permission to use the material.
The ad, called 'Bad News', is designed to get the vote out by appealing to voters who do not want Republican candidate John McCain to win the presidential election, but may not turn up at the election booth on November 4.
At one point during the commercial, NBC host Tom Brokaw and MSNBC presenter Keith Olbermann are shown, with Olbermann announcing that McCain has "won."
The Obama campaign has refused to back down over the ad and instead has attached a disclaimer to it which reads: "NBC and MSNBC did not cooperate in the making of this video."
But the disclaimer did not go far enough for NBC, which has demanded that Obama stop using the clip altogether.
Allison Gollust, an NBC spokeswoman, said: "We have received some pushback from [the campaign]. We have engaged the Obama campaign on the legal front and demanded that they stop using the ad. To date, they have not complied. Their response was to put a tag line at the end of the ad."
The video had been viewed nearly 600,000 times after being posted on The Huffington Post, Daily Kos and other websites.
NBC and MSNBC have previously been accused by supporters of McCain of showing favouritism to Obama.
The move is not unprecedented. YouTube apparently removed a McCain campaign ad that featured CBS News anchor Katie Couric chastising Barack Obama after Couric's employer, CBS, complained.
The ad attacked Obama for saying "You can put lipstick on a pig, but it's still a pig," allegedly in reference to Republican vice-presidential candidate Sarah Palin, although the Obama camp insisted the comment was about the economy.
The ad controversy comes as both candidates launch new ads focusing on the economy. In Obama's latest he says "jump-start our economy with a middle-class tax cut paid for by closing special interest loopholes".
In the latest Republican spot, Palin is given the lead and talks about the economic impact of Obama's plans to increase taxes.
"How are you going to be better off if our opponent adds a massive tax burden to the American economy?" asks Palin in the McCain ad.
Watch the latest campaign ads and read about the media issues surrounding the US presidential election in Electioneering Stateside.