The rally comes after President Aleksandar Vucic told The Financial Times this month that Serbia could begin mining lithium as early as 2028 following new guarantees from Anglo-Australian mining giant Rio Tinto.
The future of the vast mineral deposits, to be mined by Rio Tinto near Loznica, remains a perennial political fault line in the Balkan country.
It pits festering distrust in the Serbian government against Europe's plans for a greener future.
Opposition politicians were among those attending the protest, which was organised by an alliance of environmental groups.
"We have come to free ourselves," said one protester, 44-year-old mechanic Petar Cergic.
"I believe that the groundwater will be contaminated. I don't need to listen to anyone, no scientist: none of the mining here is ecological, so this cannot be either."
Another protester, 70-year-old Ljubinka Spasic denounced the mining project as a "deprivation of the right to life and existence.
"This has crossed every boundary of destruction and endangerment of Serbia and the Serbian people."
- Public health fears -
Billions of euros are at stake, with Rio Tinto saying it would provide thousands of jobs and secure Serbia's position in emerging energy markets.
The company said the area holds one of Europe's largest reserves of lithium, a strategically valuable metal crucial for electric vehicle battery production.
The deposits were discovered in 2004 but the Serbian government halted the mining project in 2022 after weeks of protests sparked by fears over the environment and public health.
"We demand that a final decision be made and that the project be terminated," one activist, Zlatko Kokanovic, told AFP as protestors began massing in the area near Loznica for the evening rally.
The project "would leave us literally without quality drinking water, lead to air pollution, and naturally, soil contamination", he added.
Vucic said the mine was expected to produce 58,000 tonnes of lithium a year, enough to supply 1.1 million vehicles or 17 percent of Europe's electric vehicle production.
Opponents have accused both Vucic and Rio Tinto of not being transparent about the process.
The mining giant recently published an environmental impact report aimed at assuaging concerns, saying the venture would rely on "safe, reliable, and proven technology".
Among the demonstrators were around thirty activists who had arrived on foot from Belgrade, more than 100 kilometres (62 miles) away.
"We wanted to show them our determination by coming to the protest in Loznica on foot from Belgrade," said Ivan Bjelic, an activist from the Mars sa Drine movement.
That he said, both the company and Serbia would "think carefully about what they will do -- and what we will do if they plan to bring machines to Loznica... or any surrounding village", he added.
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