In the latest setback to the long-running saga involving the 400-million-euro ($448 million) redevelopment of the site in the plush western sector of Paris, the Tribunal de Grande Instance (TGI) ordered an interim suspension.
It came just three days after a different court had given the green light.
On Monday, the French Conseil d'Etat (Council of State) overturned a March ruling that blocked development of a new 5,000-seat stadium in the Serres d'Auteuil due to protests over the impact the work would have on the botanical garden's historic greenhouses which date back to the 19th century.
"Our goal is to block the works. If everything is destroyed, our actions have no sense," Philippe Zagury, the lawyer of the Formige descendents, told AFP.
Since Monday, work had already started on some demolition as well as tree-felling at the Serres d'Auteuil.
Part of the redevelopment of Roland Garros will also see a roof built on the central Court Philippe Chatrier but that is not expected to be finished before 2020.
The French Tennis Federation reacted angrily to the latest delay claiming in a statement the decision was taken "in dubious circumstances" and demanded an annulment of the latest legal decision.
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