The legal complaint was filed in Vienna by Austrian environmental group Global 2000, which spearheads the movement against Monsanto and the European Food Safety Agency (EFSA).
"There are suspicions that EU authorities and the industry tried on the basis of incorrect data analysis to keep glyphosate in the European market despite its likely carcinogenic effects on humans," said Global 2000's lawyer, Josef Unterweger.
The complaint came days before representatives of the EU's 28 members will vote on March 7 and 8 on whether to continue to allow the use of glyphosate.
First used in the 1970s as the main ingredient in Monsanto's herbicide Roundup, it is now one of the world's most popular weedkillers.
But in more recent times its usage has sparked controversy, in particular after the UN's International Agency for Research on Cancer (IARC) said last March that Roundup and similar products were "probably" carcinogenic.
However, the EFSA -- which advises the European Commission, the EU's executive body -- found in November 2015 that glyphosate was "unlikely" to cause cancer.
The report prompted 96 prominent scientists, including from the IARC, to send a letter to the Commission, warning that EFSA report was "not credible because it is not supported by the evidence".
Despite the criticism, the European Commission last week revealed it would support an extension of glyphosate's license, due to expire in June, based on the EFSA's recommendation.
Global 2000 and other European NGOs on Wednesday called on EU members to "vote against an extension of glyphosate".
They accuse the EFSA of basing its findings entirely on a study provided by the German food safety authority (BfR), which in turn "obtained its data directly from Monsanto".
As a result, Global 2000 said it was also suing the German watchdog.
"Our analysis revealed serious breaches against the guidelines of cancer data analysis, which show the discrepancy between the (IARC) on the one hand, and Monsanto, BfR and EFSA on the other," said Global 2000 spokesman Helmut Burtscher.
"Authorities have to act in the public interest... This has not been the case for glyphosate."