The Bridge International Academies group says it offers affordable, high-quality education to its 12,000 pupils, who often come from poor families.
The Education Ministry says the 63 schools must now close immediately.
The group, supported by foundations such as those set up by Mark Zuckerberg and Bill Gates, says it will appeal.
A judge said the Bridge International school authorities had been given several opportunities to meet the national standards but had failed to do so.
The US-owned group first opened its schools in Uganda in 2015, but has been plagued with accusations of poor sanitation, inadequate infrastructure and not following the national curriculum, reports the BBC Africa's Catherine Byaruhanga from the capital, Kampala.
The government ordered their closure in July also alleging that the schools were recruiting unqualified teachers.
However, the organisation decided to go to court to fight the decision.
It insists it follows Uganda's public education system, with seven years of primary school and children starting from the age of six.
Some people who support Bridge International argue that government schools are worse off with nearly 70% of children dropping out before they finish primary education, our correspondent says.
Teacher absenteeism is also said to be a major challenge with about one third of teachers not turning up during the school week, she says.
Sources: Ministry of Education, 2014, Uganda National Examinations Board, 2015, Uwezo,  2015
Bridge International Academies has major backers, including the UK government and the foundations of philanthropic billionaires Bill Gates, the Microsoft founder, and Facebook's Mark Zuckerberg.
It also operates in India and Nigeria, and in January entered into a partnership with the Liberian government to run its primary schools.
How Bridge International operates in Uganda:
Read more: Is private education the answer?
Uganda's High Court has ordered the closure of a chain of private schools over concerns about poor sanitation and its curriculum.