The group of women in their 50s had set off from Nantwich, Cheshire, for a pre-booked tour of the palace, carrying bouquets which it was understood they intended to lay at the gates.
But two were seriously injured and later died when their 53-seater coach was involved in a crash with a heavy goods vehicle and a Ford Transit van near Cannock, Staffordshire.
The accident happened as Northamptonshire police and the Althorp Estate appealed for people to stop bringing flowers to Diana's family home amid fears for safety in the narrow lanes.
After Earl Spencer allowed himself to be pictured surrounded by a sea of flowers on the island where the princess is buried, the estate found even more were left at the gates yesterday.
An estate spokeswoman said they had not decided what would happen to the new blooms but they could not be taken to the island.
"It is turning into a problem," she said. "We are now concerned for public safety, both near the gates and in the surrounding lanes."
She suggested people should give a donation to the Diana memorial fund instead. Northamptonshire police backed the idea.
The committee which is examining suitable memorials for Diana is to consider the permanent closure of the Mall which links Trafalgar Square with Buckingham Palace.
The Mall has been closed since the day of Diana's death and cannot be reopened until the numbers of people visiting the St James's and Buckingham palaces drops significantly.
Officials at the Department for Culture, Media and Sport have been impressed by the fact that despite the closure of the road, extra traffic has not clogged the alternative routes.
The "Diana" committee will meet as soon as Gordon Brown, who is to chair it, returns from devolution duty in Scotland. The Department is anxious to press on with arrangements to create a permanent memorial, probably in Kensington Gardens. The task of removing the flowers will start at St James's Palace tomorrow morning to be followed by Kensington Gardens where the pile is at some points 5ft deep with the bottom layer starting to compost at a temperature of up to 180 degrees Fahrenheit.
The Department refuses to issue an estimate of the cost of the operation but with huge screens, 2,000 people and 100 contractors and sub-contractors involved, the bill is likely to betens of millions of pounds. It will be met by the Government's contingencies fund.
Thousands are still flocking to pay their respects. Up to 700 people an hour were signing the 42 books of condolence, now at Kensington Palace. The total has passed the half million mark. American Dr Jean Ford, 51, an education lecturer, flew to Britain from Chicago, USA, yesterday to "stand with the British people at this time of grief".
The BBC and PolyGram, the record company, hope to release an album of Diana's funeral service at Westminster Abbey if the Spencer family give consent.Reuse content