The activists from Mother Nature, one of Cambodia's few environmental advocacy groups, denied charges of plotting against the state, which they said were politically motivated.
The five activists were among 10 environmentalists sentenced to between six and eight years in jail last year.
Path Raksmey, 34, wife of activist Thun Ratha, told AFP that she was disappointed with Wednesday's ruling by the Supreme Court.
"I am very regretful that the court does not allow bail for them. They are the ones who protect the environment but they are locked in jail while people who have destroyed natural resources live happily," said Path Raksmey.
"It is unjust for the five," she said, adding that her husband remains "strong".
The five activists have been jailed in different prisons after their sentencing in July, but have launched appeals.
The United Nations Human Rights Office said last year it was "gravely concerned by the conviction and harsh sentencing".
The tussle over protecting or exploiting Cambodia's natural resources has long been a contentious issue in the Southeast Asian nation, with environmentalists threatened, arrested and even killed in the past decade.
Cambodian journalist Chhoeung Chheung died in December after he was shot while investigating illegal logging in the country's northwest.
Unchecked illicit logging has contributed to a sharp drop in Cambodia's forest cover over the years, according to activists.
From 2002 to 2023, a third of Cambodia's humid primary forests -- some of the world's most biodiverse and a key carbon sink -- were lost, according to monitoring site Global Forest Watch.
Cambodia's government has approved plans for a cement factory deep inside a protected wildlife sanctuary, according to an order seen by AFP on Tuesday.
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