This is equal to 4.12 percent of the electorate, and as it got more than 100,000 signatures the question of a referendum must now be discussed in parliament.
The petition was initiated by 66-year-old Inge Rauscher, who launched a similar petition back in 2000 which was only signed by 3.35 percent of the electorate.
The retired translator has been against EU membership ever since Austria joined the union.
She and her supporters believe that Austria would be better off without EU membership - economically, socially and environmentally.
The petition gathered the strongest support in Lower Austria (5.18 percent) and in Carinthia (4.85 percent).
Rauscher said that she believes there was more support for a referendum this time around due to the economic downturn and the Greek crisis. In a press release she said it was “a great result”.