The advertisers, along with other members of the Association of National Advertisers, have sent a letter to the Justice Department asking it to block the agreement as bad for advertisers.
The deal between Google and Yahoo! combines their search advertising systems and will see Google Ads appear on Yahoo! search engine results in the US and Canada.
It has been estimated that it could boost Yahoo!'s revenues by up to $800m as its struggling chief executive Jerry Yang casts around for a way to block Microsoft's $44.6bn (£22.6bn) takeover bid.
In a statement on its website the Association of National Advertisers said prior to sending the letter to Thomas Barnett, assistant attorney general, US Department of Justice it conducted a comprehensive, independent analysis, which included input from the board's members and face-to-face discussions with Google and Yahoo!.
The ANA said it was concerned that a Google-Yahoo! partnership will control 90% of search advertising inventory and the partnership will likely diminish competition, increase concentration of market power, limit choices currently available and potentially raise prices to advertisers for high quality, affordable search advertising.
The letter comes as the Department of Justice looks at the search deal and it will increase pressure on Google, which prior to this managed to avoid confrontation with advertisers who have been worrying about causing friction with the search giant because of its power in the digital marketplace.
A report in the Wall Street Journal says the deal has been endorsed by advertising agencies and midsize advertisers.
Bob Liodice, chief executive of the ANA, told the paper that on balance the deal is a negative one for advertisers.
Yahoo! said that it was "disappointed" with the ANA's decision and Google said that the deal had been supported by numerous advertisers, but did not immediately name any.
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