Residents in Camden, north London, unsuccessfully campaigned to save the site at St James' Gardens, which was used as a burial ground from 1790 to 1853.
HS2 Ltd will excavate sections of the ground to enable the removal of the remains which will be re-interred elsewhere.
Campaigners have strongly opposed the construction work for the £55.7 billion rail scheme, including the cutting down of more than 500 trees in the borough.
Dorothea Hackman, a church warden who helped to organise the private memorial service, said 40 people were allowed to gather at the now closed St James' Gardens to pay respects to the people buried there.
"Some local people were very upset," she told the Press Association.
"People have been buried there for an incredibly long time. It's been very moving.
"But this is also about the living, and people being able to mourn the trees. We're incredibly upset about the trees.
"This will have a massively negative impact on the health of the community and the children growing up here for the next 17 years.
"I would be delighted if the Treasury would decide that the economic case for HS2 hasn't been proven."
Local resident Marian Kamlish, 92, said: "In times of austerity, this vanity project is an insult to those in the NHS, in education, in the fire services and the police force, all suffering cuts while this rich man's railway sucks up our money."
Several notable people are buried at St James' Gardens, including Lord George Gordon, associated with the 1779 Gordon riots; Captain Matthew Flinders, the first person to circumnavigate and name Australia; and Bill Richmond, a pioneering black boxer.
An HS2 spokesman said: "Though the former burial ground at St James' Gardens has not been in use for more than 100 years, we will ensure that we treat the site with dignity, respect and care.
"As such, we will continue to work closely with the local community, the Archbishops' Council, the local parish, Historic England and other organisations as we proceed with the next phase of the project."
He added that the work at Euston will triple the number of train seats out of the station at peak hours and create a "gateway to the capital and the nation" that the local community and travelling public can "rightly call their own".
In February, Parliament granted powers to build Phase 1 of the line, which is due to open in December 2026.
This will see trains travel at high speed between Euston and Birmingham before running on from Birmingham on the existing West Coast Main Line.
Preparatory work has begun and major construction projects are due to launch in 2018/19.
Services are expected to operate on Phase 2a from the West Midlands to Crewe in 2027, with Phase 2b from Crewe to Manchester, and Birmingham to the East Midlands and Leeds, due to open in 2033.
HS2 Ltd said all removed trees will be replaced with a mixture of native species "reflecting those that we lose", and it is working with Camden Council to identify suitable locations. Planting will begin later this year.
Listen to Dorothea Hackman speaking to Premier's Eno Adeogun: