Others said the project should be allowed to move through the permitting phase before any action is taken that could curtail development.
Tom Collier, CEO of the Pebble Limited Partnership, which is working to advance the project, said it was "ludicrous" to hold a public meeting so soon after releasing a lengthy technical report.
"This hearing is much more about show than it is about substance," he said.
EPA's review of whether to impose restrictions on development, through a rarely invoked process under the federal Clean Water Act, could take a year. The agency's regional administrator, Dennis McLerran, said he was taking notes from the testimony and said the proposal could be modified.
Pebble supporters, though, fear the EPA is on track to pre-emptively veto the project.
The Pebble Partnership and state of Alaska have sued, saying the EPA overstepped its authority. The agency has asked a federal judge to toss the case.
At Tuesday's hearing, tribal leaders, religious leaders, fishermen and environmentalists spoke in support of the EPA.
Mary Ann Johnson, who grew up in Naknek on Bristol Bay, said village residents can't depend on the state to look out for them.
Several legislators also testified, some in support of the EPA's actions, some against.
"I'm just a working stiff like everybody else around here. I'm not a lawyer," said Rep. Pete Higgins, R-Fairbanks, a dentist by trade. "This is not about tribes. This is not about Pebble mine. This is about government overreach."
The Pebble deposit is on state land.
Deantha Crockett, executive director of the Alaska Miners Association, said she regularly takes calls from potential financiers wondering if investing in mining in Alaska is a safe bet. "And I don't know what to say to them," she said.
Everett Thompson, a commercial fisherman from Naknek and Bristol Bay Native Corp. shareholder, urged the EPA to stay tough.