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."

The new reporting system, called 'Step Up', is in use at Ballyclare High School and can be used at home, in school or on a smartphone.
The school believes it is the first system of its kind in Northern Ireland.
It means pupils can raise concerns with the appropriate teacher directly via the school website.
14-year-old Emma Blacoe, who helped develop it, said that teachers could receive a lot of information about alleged incidents.
"Pupils can upload files and pictures of screenshots and chats if they feel someone's in danger," she said.
"They can also simply just say if they're being pressured or bullied or if anyone else is."
If pupils click on a button on the school website they are taken to an online form where they specify their year group and who they want to deal with their concerns.
The information they provide cannot be seen by anyone but themselves and teaching staff, and they can choose to give their name or remain anonymous.
David Andrew, who also worked on 'Step Up', said that it was easy to operate and accessible to pupils.
"If pupils really feel there's something which needs to be sorted out they can do it," he said.
"Bullying is never acceptable and the sooner someone steps up, the sooner it can be sorted out."
The school's head of cyber safety, Mhairi Hill, said teachers would investigate every allegation they received information about.
"Staff will pick it up and the earliest opportunity and follow the procedure already in place for dealing with incidents," she said.
"With online bullying, pupils can come to us with screenshots and that's concrete evidence we can use to take the investigation forward.
"We would establish the facts before proceeding but we would follow up every report.
"If a child has felt it's necessary to raise the concern then it's worth following up in our eyes.
"We have a duty of care for our pupils and if a pupil's upset or distressed in class they're not going to learn to their full potential so it's important that we address these issues."
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".
[NEXT_CONCEPT]
Pupils at a County Antrim school have developed a new tool to target cyberbullying.