The Botswana High Court said on Wednesday the Bushmen people were not entitled to use a well already established on their traditional land in the Kalahari Game Reserve or excavate a new one.
The government has argued that the Bushmen's presence in the reserve is not compatible with preserving wildlife and that living in such harsh conditions offers few prospects.
In 2006, another court allowed the Bushmen to return to desert-like homelands where diamond mining claims and a new luxury tourist lodge led to their eviction by the government.
Hundreds returned and their leaders protested that they were denied water in a scheme to drive them away again.
After the ruling on Wednesday, community spokesman Jumanda Gakelebone said they will seek legal advice to fight the ruling.
"It's a sad day," he said. "If we don't have water, how are we expected to live?"
He told The Associated Press in a telephone interview that a tourist lodge with a swimming pool and a bar and wells at diamond claims lay just a few kilometres from Bushmen settlements.
He said watering holes for wild animals were paid for by diamond and tourist firms, but Bushmen were prevented from carrying water into the game park for their families.
Survival International, an international support group for indigenous peoples, immediately condemned the ruling in the southern African country.
"In the last 10 years, Botswana has become one of the harshest places in the world for indigenous peoples. If Bushmen are to be denied water on their lands when it is freely provided for tourists, animals, and diamond mines, then foreigners should be asked if they really want to support this regime with their visits and jewellery shopping," said group director Stephen Corry in a statement.
The Bushmen's water case was heard in the Botswana High Court in early June, but judgment was reserved until Wednesday.
Gakelebone said the government first sealed a well used by the Bushmen in 2002 to drive them off their land to make way for tourism and mining.
Community leaders won their right to return on constitutional grounds in the 2006 case, but they were still prevented from reopening the old well, he said.
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