"Wooden barriers have to be taken down and women have to be allowed to join, to pray behind the men in the main praying area. That's our request," said Fatima Thompson, an American Muslim who converted to the faith 18 years ago.
"We are against gender segregation, against the fact that women are put aside or in a totally different room at the mosque," added Thompson, who led the group of self-identified progressive Muslims.
Urging for the right to pray together with the male members, Thompson said that the discrimination leaves a bad impression about the religion.
"The general issue we are pushing is gender segregation and the ramifications it fosters. It's not healthy, and not reflective of our society here. It's very reflective of very restrictive, ultra orthodox societies," Thompson added.
A group of six women in headscarves walked into the Washington mosque, situated in city's embassy district, through the main door and entered the praying area which is reserved for men, much to the surprise of around 20 men who had already arrived there.
Ordinarily women and chidren enter the mosque through a small door hidden behind a screen.
This is the second incident in which women had sought the right to pray in the main area at the mosque in Washington DC.
In Feb 2010, a group of 20 women carried out a similar protest.