Almost 5,000 people signed a petition in a bid to protect The Brimmon Oak, Newtown, in the path of the bypass.
The Welsh government has agreed to adjust the route to accommodate the oak with "minimal work" within a 15m (49ft) protected zone around the roots.
But opponents called for further assurances in a letter to the assembly petitions committee on Tuesday.
"We're not against the bypass, we just want them to move it over a bit," said campaigner Rob McBride.
Mervyn Jones, who owns the land the tree sits on, believes the roots will be damaged and it will die if the road is not far enough away.
The committee agreed to pass along the petitioners' concerns to the government.
Ministers have agreed to route a planned bypass around a 500-year-old oak tree in Powys.