Companies moving into part of the Markham Vale business area, off Junction 29A of the M1, want to see a current service extended to the top of Seymour Link Road.
Firms say they are desperate to fill vacancies but are unable to attract employees because of the lack of public transport.
A study of the barriers stopping people working at the site, undertaken by businesses, has found a lack of buses to be significant.
Derbyshire County Council says it does not currently have the budget to fund more services – but has promised to investigate the issue further.
Labour councillor Anne-Frances Hayes, who is backing the campaign, said: “I believe the extended route will assist businesses in recruiting to staff vacancies – and, in particular, will help the recruitment of local people to work at companies in Markham Vale.
"In addition, an improved bus service will benefit the environment by reducing the number of existing single occupancy vehicles, conserve fuel, decrease air pollution and reduce the region’s carbon footprint.”
The calls have also been backed by leading recruitment agencies.
Izabella Edwards, managing director of Edwards Employment Solutions, said: “As a business community we must start the conversation about improving transport links to all of Chesterfield’s industrial areas, engage with Derbyshire County Council and work towards solutions.
"The current provision is inadequate and is a barrier to a large number of workers accessing opportunities to work – often young and disadvantaged people.”
Tom Cottingham, from The Best Connection agency, said workers have ‘expressed frustration’ in there being no bus routes to the newly-built estate.
"They want to do the job but rely on public transport and are having to turn down opportunities,” Tom said.
"It is very frustrating for us. The aim is getting people into work and the council should be supporting that.”
He added six new companies had moved in the industrial estate but without a bus route in operation people cannot apply to work there.
A Derbyshire County Council spokesperson said: “We currently fund service 81 and the evening and Sunday service 1 into Markham Vale.
"Unfortunately an extension of the current route to include the Seymour Link Road is logistically not possible.”
She added: “This position may well change soon however, and it is possible that improvements to the current services could be looked at as part of implementing the council’s Bus Service Improvement Plan over the next three years.
"We’d welcome further contact from the businesses.
"If they could provide us with details of shift patterns and the areas where bus services need to be provided from then this would help us to investigate further.”