Residents fear that the Cerro Blanco site, which has been granted permission by Guatemala to convert from an underground to open-pit mine, will contaminate Lake Guija.
"All these extraction projects do is sell out our environment and our future," said Claudia Rodriguez, a 41-year-old member of an association of women environment defenders.
"The goal is for Cerro Blanco to close, even if we have to give our lives for it," she said.
The previous Guatemalan government in January gave approval to Canadian owner Bluestone Resources to turn Cerro Blanco into an open-pit mine, just days before right-wing president Alejandro Giammattei left office.
The government of El Salvador expressed "serious concern" about the move.
On Thursday, Guatemala's new government said it was seeking to reverse the decision due to what it called "anomalies."
Opponents say the site poses a serious threat to the environment.
"The use of chemicals in mineral extraction is greatly damaging our natural resources and the Cerro Blanco mine is no exception," Salvadoran environmental campaigner Ricardo Navarro said.
"The use of chemicals in that mine will harm not only El Salvador, but also Guatemala and Honduras," he told AFP.
Chemicals harmful to humans, animals and plants would pollute the Lempa river that flows through the three countries, Navarro said.
Lake Guija feeds a tributary of the Lempa River, which starts in Guatemala, crosses part of Honduras and then enters El Salvador, where it is an important source of drinking water for the capital San Salvador.
Videlina Morales, a 56-year-old anti-mining activist, said she feared for the future of the lake, which local fishermen rely on to earn a living.
"We demand the closure of the Cerro Blanco mine out of respect for our natural resources," she said.
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