NewsCorp, which is owned by Rupert Murdoch, gave $1 million (£632,000) to the United States Chamber of Commerce, a business group that campaigns against Democratic candidates.
In August, the company donated $1 million (£632,000) to the Republican Governors Association - one of the biggest ever political gifts from a media organisation.
The chamber of commerce has said it would spend about $75 million (£47 million) on campaigning for next month's mid-term elections. The vast majority will be used to oppose Democrats.
News Corporation has previously said it donates to organisations "which have a pro-business agenda, [and] support our priorities at this most critical time for our economy".
In an interview published this week Barack Obama criticised Fox News, whose broadcasters are often sharply critical of his presidency.
Mr Obama told Rolling Stone magazine that the channel's viewpoint was "ultimately destructive for the long-term growth of a country that has a vibrant middle class and is competitive in the world."
Mr Murdoch defended the channel in evidence to a congressional committee on Thursday. He said: "We welcome all views on Fox News. We don't censor." The Australian media mogul was speaking in a House Judiciary Committee hearing on immigration. He and Michael Bloomberg, the billionaire mayor of New York, explained why they support pro-immigration reforms.
Earlier this month, Mr Murdoch personally donated $1,500 to the California gubernatorial campaign of Meg Whitman, the Republican candidate and former chief executive of eBay. Mrs Whitman is currently under intense political pressure after it emerged that her housekeeper was an illegal immigrant.