Prosecutors say Nikolaos Frangos, part-owner of Liberty, and George Capuzello, the project foreman, ordered their employees to ignore restrictions on the work site meant to protect the falcon nest.
As a result, workers with heavy construction equipment scared off the birds, which abandoned their unhatched eggs, investigators from the U.S. Fish and Wildlife Service said.
Once the damage was done, authorities say, Frangos and Capuzello lied to investigators about the unauthorized work in an attempt to cover up the fact that they had several undocumented immigrants working on the site.
One of those men - Mikhail Zubialevich, known on the work crew as "Russian Mike" - was charged along with his bosses with making false statements to investigators. He pleaded not guilty during an initial appearance in federal court Wednesday.
Another worker, Walter Morgan, pleaded guilty in February to taking or destroying a migratory bird egg.
Frangos' lawyer, William DeStefano, said Wednesday he did not understand why prosecutors had targeted his client. "These guys paint bridges," he said. "It's not the case of the century."
The Liberty-Alpha joint venture won a $70 million contract from the Pennsylvania Department of Transportation to repair the bridge in 2009.
As part of the deal, the firm was required to work around the falcon nesting season and employ a dedicated environmental expert to ensure the birds' safety.
Peregrine falcons were removed from the federal list of endangered species in 1999.
Inquirer staff writer Sandy Bauers contributed to this article.