Nicholas Burgess, a boss at LCP Properties which owns the Dunstall Hill Trading Estate on Gorsebrook Road, said the area was "not appropriate" for a travellers site and that tenants on the industrial estate had already raised concerns about the potential impact on trade.
Mr Burgess said he believed the council would need to go through his industrial estate to access the land which is the size of a football pitch and currently covered by trees and if that was the case he would refuse.
It is the latest development in a deepening row over the proposals which have sparked a fierce backlash locally.
It came following a public meeting held over the issue at the Holiday Inn at Wolverhampton Racecourse last night.
Mr Burgess, managing director of LCP Properties, said: "No industrial estate relishes the thought of having a travellers site next door.
"If they needed access across our land I would refuse it. We own most of the industrial estate and the road leading up to it.
"They have said that particular piece of land was to be used for some bio-diversity reasons.
Trees would be lost there that also act as a buffer between the park and the industrial estate.
"Our tenants and people on the estate have contacted me to state this really shouldn't happen."
The complaints from businesses come on the back of a backlash from residents living nearby, who have signed a petition and penned an open letter calling for support from local councillors.
Wolverhampton Council says it must provide a legal site for travellers to pitch up if they are to retain powers to protect 60 public spaces in the city, which could otherwise be left vulnerable.
Mr Burgess said he would be prepared to state his objection to the plan if asked to by businesses.
He added: "If the majority of tenants ask me to submit recommendations I think I would on behalf of those whose livelihoods may be disrupted and affected."
A Wolverhampton Council spokesman said: “We understand some residents and businesses may have concerns about the proposed plans for the site.
“Our feasibility studies have highlighted a number of options to access the site.
"We would prefer to use the established access but have identified several alternatives utilising council-owned land adjacent to the highway, if we cannot reach an agreement with the landowner.
“We are taking all comments on board and happy to meet with the land owner to discuss any concerns.
“This is why we are consulting with the public and holding our public meeting to ensure the voices of our residents and business owners are heard before any decisions are finalised.”