The RSPB has questioned whether HS2 carried out proper surveys “before sending in the chainsaws”.
And activists and local householders in Buckinghamshire, who have condemned the “ecocide”, are planning a legal challenge to the tree felling.
At the same time, the Wildlife Trusts announced that HS2 had taken possession of a lake in Middlesex, “a stunning wetland that is home to precious wintering wildfowl”.
The Buckinghamshire trees, described as irreplaceable, are close to residents’ homes in Leather Lane, near Great Missenden.
Hedgerows and trees nearby in the Chilterns have already been cleared in preparation for the railway line from London to Manchester and Leeds, which is forecast to cost £106bn.
Jim Ashton, a wildlife expert and conservationist, whose land borders the area of oaks and other trees, posted a video showing workers removing bird boxes. He said he had previously filmed blue tits using one, and the other contained nesting materials.
Campaigners claim it could be evidence of a crime, because the Wildlife and Countryside Act 1981 makes it an offence to damage active nests.
But HS2 says protesters placed the bird boxes there to lure birds close to the works site.
“I have spent the last 18 months observing and documenting the diverse ecology in and around these majestic oaks, from roosting bats, to nesting blue tits, tawny owls, breeding tree creepers and a resident kestrel,” said Mr Ashton.
“To me, it is inconceivable that work can continue. It’s a scandal beyond words.
In a video at the scene while tree felling was under way, he added: “I feel sick. We can’t sleep. I can’t believe I’m in the UK. As long as I live, I’ll be traumatised by this.”
Pointing to a tree being pulled down that he said had blue tits in, he said: “This ecocide is just disgusting on every scale. It’s heartbreaking.”
He told The Independent that in the past 18 months he had seen bats many times at dusk “whizzing up and down among the trees” and it was “inconceivable” that they would not have roosted in the trees.
RSPB England tweeted: “Did you do the surveys @HS2Ltd before sending in the chainsaws?”
Mr Ashton also said he had documented potential bat roosts in the trees, posting photographs of the holes.
According to HS2 Rebellion, any hibernating bats would probably be killed. The group said an independent ecologist had found a bat roosting feature with “possible signs of use including scratch marks and smoothed edges where bats naturally exit”.
But the ecologist concluded that it was impossible to say whether the roosts were inhabited before May, when bats become active.
“We have received reports claiming that survey efforts at this site have been too little and at the wrong time of year,” the group says.
Residents are also angry at the destruction. A petition says they fear “the nature of this ancient lane will be completely destroyed in a matter of weeks”.
The Berks, Bucks and Oxon Wildlife Trust wrote: “The government needs to stop and rethink. Detailed surveys should be done before any felling takes place. If HS2 has not done this and active nests are damaged, the matter should be reported to the police.”
The Wildlife Trusts said the work at Broadwater Lake in Middlesex would “carve the site in two and directly harm and disturb wildlife”.
HS2 insists all its ecology work is carried out in accordance with the law.
A spokeswoman said bird boxes had been put up by protesters to try to get birds to nest close to the work, “putting the birds in danger rather than protecting wildlife”.
She added: “We take our environmental responsibilities seriously and we conduct ecology surveys ahead of the start of permanent works. We have worked hard to reduce the number of trees lost at Leather Lane and no trees will be removed just for temporary works.”
“We are committed to only remove vegetation that is necessary to construct and safely maintain the railway.
“There is no evidence of bat roosts at Leather Lane.”
Protesters deny the bird boxes were put in place deliberately on the HS2 site.
HS2 says 7 million trees and shrubs will be planted between London and Birmingham, and 60 wildlife habitats have been created along the route.
Of the Middlesex lake, a spokesperson said: “We have taken possession of a small section of Broadwater Lake, but our works won’t cause any severance. A viaduct will be constructed to the southwest – there is no contact with the lake. Our works have temporarily reduced access along the western side of lake and we’re currently working up plans to provide pedestrian access.”