The programme to keep leaves and branches off the lines has already involved the destruction of thousands of horse chestnuts, ash, poplar and sycamore trees.
But the RSPB last night warned that such activity should only take place at this time of year – the nesting season – if trains were at imminent risk.
The warning follows a row in Bournemouth after the British Transport Police was called following complaints Network Rail was felling hundreds of trees along the railway line, prompting concerns about nesting birds.
Meanwhile felling along the trans-Pennine route from Manchester to Leeds was described by one passenger as resembling a “logging operation”.
Network Rail has previously said the green corridor along its tracks acts as a haven for wildlife.
An RSPB spokesman said: “Tree pruning and felling should be avoided at this time of year to avoid harming birds.
“If it is emergency work for urgent safety reasons, then, of course, it needs to be done, but the worry is that much of this work is simply routine, non-urgent work that is simply being carried out with little regard to the presence of birds and other animals.
“If such routine work is being done without reference to the Wildlife and Countryside Act, which offers basic protection to nesting birds, it may well be in breach of the law.”
Network Rail said its operatives were adhering to “all environmental requirements”, including daily checks for nesting birds.
“Managing vegetation is vital to running a safe and punctual railway,” a spokesman said.
“Getting everyone home safe every day is our top priority.”
Network Rail has said the Bournemouth work was justified by “safety issues”.