The EU has progressively clamped down on animal testing since the 1990's and banned most such products in 2009, but it left a few exemptions for several toxicity tests which will now cease.
The ban applies to all products, wherever in the world they come from.
The European Commission has "thoroughly assessed the impacts of the marketing ban and considers that there are overriding reasons to implement it", it said in a statement on Monday.
"This is in line with what many European citizens believe firmly: that the development of cosmetics does not warrant animal testing."
EU Health Commissioner Tonia Borg said Brussels would continue "supporting the development of alternative methods and to engage with third countries to follow our European approach".