The television ad for the Democratic congressman from Memphis, who is running against former Chattanooga Mayor Bob Corker, a Republican, shows Ford in a church talking about his religion and values.
The two men are vying for the seat of retiring Senate Majority Leader Bill Frist of Tennessee.
"To our knowledge, this is the first time a partisan political ad has been produced using the backdrop of a church," said Ellen Johnson, president of American Atheists, in a statement. "It's part of a larger and disturbing trend where candidates are invoking religion in order to woo constituencies and win elections."
The Ford campaign had a quick answer to the criticism.
"Being opposed by (American Atheists) makes us even more certain that Harold Ford Jr.'s Senate campaign is being embraced by people of faith," said Michael Powell, senior adviser to the Ford campaign.
Johnson said that "playing the religion card" excludes millions of atheists, freethinkers, secular humanists and other nonbelievers.
The ad was filmed in Mount Moriah-East Baptist Church in southeast Memphis.
Jeremy Leaming, a spokesmand for Americans United for Separation of Church and State was quoted in one article as saying, "It sounds problematic for a house of worship to open its doors to what appears to be blatant campaigning. I also think politicians should respect houses of worship and not try to drag them into politicking."
It is not clear whether the church could be charged with violation of the Internal Revenue Service laws that prohibit tax-exempt groups, such as churches, from involving themselves in campaigns.