Edward Wytkind, of the Transportation Trades Department, a labor union, called Bunning's move "irresponsible" and "downright dangerous."
"In this economy, to purposefully put people out of work is cold-hearted," he said. "It's even worse that these workers perform essential functions to expand and build our nation's transportation system and ensure it is safe for all its users."
"He's preventing the Senate from moving forward," said Vice President Joe Biden today. "400,000 people will be kicked off the rolls this month if he has his way."
Senators will take up a more long-term version of the bill to extend unemployment benefits on the Senate floor today, but it's not likely to pass until later this week or next week.
As for the highway trust fund, money for that program is likely to pass the House this week as part of a jobs bill already passed through the Senate, meaning furloughed workers could soon be back to work.
Bunning, who has served in the Senate for over a decade, has a history of saying inflammatory things. During his 2004 re-election campaign, Bunning described his Democratic challenger as looking "like one of Saddam Hussein's sons." After Justice Ruth Bader Ginsburg announced she had pancreatic cancer, Bunning said she would "likely be dead in nine months." He later apologized. More recently, Bunning threatened to sue the National Republican Senatorial Committee if it fielded a challenger to him in the primary.
ABC News' Lisa Stark and Matt Loffman contributed to this report.