WASHINGTON — Until just a few months ago, Evan Bayh was working for the U.S. Chamber of Commerce. Now the chamber is working as hard as it can against him.

In a campaign twist with significance in the battle for the Senate, the nation’s leading business lobby is going all out to defeat Mr. Bayh, the former Democratic senator from Indiana trying to reclaim his old seat. The chamber is doing so despite the fact that Mr. Bayh, considered a business-friendly centrist, was on the chamber payroll as recently as June and made appearances on the organization’s behalf after leaving the Senate in 2011.

The chamber’s decision to attack its former ally has angered Senate Democrats to no end because capturing the Indiana seat is a crucial element of their plan to take back the Senate majority. Democrats’ efforts to persuade the chamber to stay out of the race as a demonstration of bipartisanship were rebuffed. The result has strained ties with Democrats, who might be chilly to future overtures from the chamber, particularly if they are running the Senate.

“The fact that the U.S. Chamber has spent millions against Evan Bayh, a pro-business candidate who has even served on some of their advisory boards, rips away any pretense of bipartisanship,” Senator Chuck Schumer of New York, in line to be Democratic leader, said in a statement. “The national headquarters is a totally partisan place and has dramatically hurt their ability to work with our caucus in 2017.”