Immigration officials are investigating whether Upper Crust Pizzeria harbored and exploited illegal immigrant workers, the second federal inquiry into the restaurant chain, according to several former employees with direct knowledge of the new investigation.

The scrutiny by Immigration and Customs Enforcement, or ICE, is the latest problem for Upper Crust, which also is contending with organized boycotts and the departure of at least three store managers following a December Boston Globe story in which some former employees alleged the company routinely took advantage of Brazilian workers by underpaying them for long workweeks.

They said Upper Crust relied on low-paid illegal laborers from a small town in Brazil to expand the chain to 18 restaurants over the past decade.

In addition to the immigration investigation, a US Department of Labor inquiry into Upper Crust’s wage practices has been underway for several months. Labor Department officials are looking into allegations that the Boston restaurant chain rescinded thousands of dollars in back overtime wages the agency required Upper Crust to pay employees in 2009.

When asked for a comment on the reported investigation, Chuck Jackson, a spokesman for Immigration and Customs Enforcement, which operates under the umbrella of the Department of Homeland Security, said, “ICE has no information available for you.’’

And David Berman, a lawyer representing Upper Crust, said the Boston chain does not know of any such investigation.

Local activists also have launched boycotts to protest what they say is Upper Crust’s mistreatment of workers.

Massachusetts Jobs With Justice, a coalition of labor, community, faith, and student organizations, is coordinating demonstrations scheduled to be held outside the chain’s restaurants over the next two weeks, according to Russ Davis, the coalition’s executive director.

“We want to help educate the public so that diners know what is going on and employers realize there is a consequence to exploiting workers,’’ Davis said.

Berman said boycotts of the restaurants are misguided. He characterized the allegations against Upper Crust as “false, malicious, and defamatory,’’ and said, “we regret that anyone has seen fit to refrain from patronizing Upper Crust’’ because of the reports.

Last month, more than 60 student groups at Harvard University joined together to boycott Upper Crust restaurants.

“We do not condone employment practices that violate the rights of workers,’’ said Claire Valentin, president of the Harvard Immigration Project. “We believe that substandard wages, denial of labor and employment rights, and the exploitation of immigrant labor undermine working conditions for all American and immigrant workers.’’

Federal immigration officials began examining Upper Crust earlier this year, according to the former workers who requested anonymity because the investigation is ongoing.