Party officials say the lights contravene traffic regulations and, at a cost of €63,000, are wasting taxpayers' money.
Toni Mahdalik, transport spokesman for the FPÖ, called the initiative gender politics "gone mad", and said the money would have been better spent on reducing poverty and unemployment figures.
Vassilakou, who is in charge of traffic issues in Vienna, says that the lights conform to laws and she hopes that the signals will draw more attention to pedestrians and reduce jaywalking and accidents.
The lights will be in use until June, and are also intended to show how open minded the Austrian capital is.
This month Vienna hosts a series of events linked to themes of tolerance - including the Eurovision Song Contest, which last year was won by Austria's "bearded lady" Conchita Wurst, and the Life Ball which is Europe's biggest charity event for Aids and HIV research.