Cameron and Tyler Winkelvoss last week agreed to drop a US supreme court action against Facebook, only to follow that up by filing a fresh lawsuit against the company.
The brothers claim that the social networking giant "intentionally or inadvertently suppressed evidence" during their previous litigation battle.
The twins agreed a $65m (£41m) settlement in 2008 after claiming that Zuckerberg stole their idea for Facebook when they hired him to code their ConnectU site in 2003. The legal battle formed the basis of hit movie The Social Network.
In January, the brothers attempted to reopen the case at the supreme court, claiming that they should have received more shares in Facebook. However, they decided to drop the action last week.
In their most recent lawsuit, filed last Thursday with the US District Court of Massachusetts, the Winklevoss twins and their fellow ConnectU founder Divya Narendra claimed that Facebook held back some crucial information during the settlement negotiations.
The filing argues that Zuckerberg failed to disclose important documents in regards to the relationship between him and the brothers while they were studying at Harvard.
Facebook's outside counsel Neel Chatterjee said in a statement: "These are old and baseless allegations that have been considered and rejected previously by the courts."
Also last week, Netflix founder and chief executive Reed Hastings joined the board of directors of Facebook, bringing his experience in digital entertainment to the firm.