It is providing legal representation for two individuals in separate cases relating to "unreasonable difficulties" and "less favourable treatment" in the provision of services on the internet.
The charity is taking the action under the Disabilities Discrimination Act 1995. Although the Act does not specifically mention web sites, section 21 of the Act relates to the provision of goods and services, which can include content on web sites.
Julie Howell, digital development officer at the RNIB, said no cases of this kind had yet been taken under the Act. "We're not naming the organisations because we want to raise awareness of the issues rather than point the finger of blame," she said.
But the British Council of Disabled People said the largest disability charities still don't do enough to champion the rights of disabled peoples.
"This is the best way that the RNIB has ever spent any of its funds," said Andy Rickell, director at the British Council of Disabled Peoples.
"Too often the big seven disabled charities are so terrified of losing their government contracts that they don't want to make too many waves and take any contentious action."