This is the principle that all websites and apps should be equally accessible.
Travel portal Cleartrip.com and media giant Times Group both announced they would be withdrawing from the service, citing competition fears.
But Facebook boss Mark Zuckerberg defended Internet.org's aims.
The service aims to extend internet services to the developing world by offering a selection of apps and websites free to consumers.
Telecoms firms absorb the data costs associated with handling Internet.org traffic.
But many companies fear this distorts the market and makes it harder for small companies to get their products seen by the public.
Currently, Indian visitors to Internet.org can access nearly 40 stripped-down services, including job, healthcare, news and education sites.
Times Group blogged that it was appealing "to all publishers to jointly withdraw from Internet.org" and said that the Times of India newspaper would also withdraw "if its direct competitors - India Today, NDTV, IBNLive, NewsHunt, and [the] BBC - also pull out".
The BBC has yet to respond to the call.
A Times Group spokesman said: "We support net neutrality because it creates a fair, level playing field for all companies - big and small - to produce the best service and offer it to consumers.
"We will lead the drive towards a neutral internet, but we need our fellow publishers and content providers to do so as well, so that the playing field continues to be level."
Cleartrip said the debate around net neutrality had given it "pause to rethink" its approach to Internet.org.
"What started off with providing a simple search service has us now concerned with influencing customer decision-making by forcing options on them, something that is against our core DNA," wrote Subramanya Sharma, Cleartrip's chief marketing officer.
In India, the issue hit the headlines this week after telecoms provider Bharti Airtel said it would allow mobile app developers to pay data charges enabling users to access the apps for free.
Start-up developers with little funding could therefore be at a commercial disadvantage, tech firms argue.
Facebook launched Internet.org in India in February after partnering with Indian telecom carrier Reliance Communications.
India has the world's third-largest internet population and is the first Asian country to get the service.
Mr Zuckerberg defended the initiative in an article for the Hindustan Times newspaper, saying: "Net neutrality is not in conflict with working to get more people connected.
"We will never prevent people accessing other services, and we will not use fast-lanes."
A group of Indian technology and internet companies have pulled out of Facebook's Internet.org initiative, fearing it threatens the principle of "net neutrality".