The £1.1bn M4 relief road around Newport would be a "direct attack on nature", conservationists have told a public inquiry into the plans.
The Gwent Wildlife Trust said the scheme would "rupture" what it calls "Wales' own Amazon rainforest".
The Gwent Levels are home to eight Sites of Special Scientific Interest and are a national nature reserve.
The Welsh Government has said mitigation work and economic benefits outweigh the environmental impact.
The government wants to build a six-lane, 14 mile (23km) motorway south of Newport between the current M4 junctions between 23A at Magor and 29 near Castleton.
"Building a motorway to bypass a motorway is like loosening your belt to fight obesity," said the spokesman for the trust at Wednesday's meeting in Newport.
"We hope that a recommendation that the scheme is not progressed will send out a strong message that Wales stands for sustainable development, not sustained development," he added.
What are the Gwent Levels?
- 125 hectares (308 acres) of Sites of Special Scientific Interest habitat, including grazing marsh and reed beds
- An area criss-crossed with ancient waterways known as reens
- Home to the UK's smallest flowering plant, rootless duckweed, which grows nowhere else in Wales
- Mammals such as otters and recently reintroduced water voles thrive on the Levels
- Breeding wading birds there include lapwing, redshank and curlew
- Cranes recently bred after being extinct in Wales for 400 years
In documents responding to objections, the Welsh Government said it accepted during construction and operation of the new road there would be "a large or very large adverse effect on the landscape character of the Levels".
But it added: "That effect has to be weighed against the significant social, economic and other environmental benefits that the scheme would bring to Newport, the wider Cardiff region and Wales as a whole."
However, that interpretation has now been questioned by the government's own future generations commissioner.
M4 relief road key points:
- The Welsh Government plans to begin construction in 2018 and open the new road in 2021
- It said the current M4 around Newport, opened in 1967, "does not meet modern motorway design standards"
- There have been 335 formal objections, compared to 192 letters of support