The Microsoft chief executive said his company had "moved on" when asked if his firm would be willing to renegotiate a deal, just a day after Google walked away from its proposed search partnership with Yahoo! due to opposition from antitrust regulators.
In May, Microsoft abandoned a $44.6bn (£28.3bn) bid for Yahoo! after Yang blocked the acquisition.
At a business conference in Sydney, Ballmer said: "We tried at one point to do a partnership around search and that didn't work either, and we moved on and they moved on.
"We are not interested in going back and re-looking at an acquisition. I don't know why they would be either, frankly."
However, Ballmer hinted that there could be opportunities for some kind of search partnership, but did not elaborate.
Yang fuelled speculation of another deal when he told industry insiders he would be willing to sell his firm to Microsoft after Yahoo!'s partnership with Google fell through on Wednesday.
Yang said Wednesday: "To this day, I would say that the best thing for Microsoft to do is to buy Yahoo!. I don't think that is a bad idea at all."
Yahoo! stock prices surged as much as 7% on rumours that Microsoft would consider another deal.
Yang is also reported to be talking with Time Warner about a combination of its AOL business with Yahoo!