Home Office data published for the first time, shows around 50 people who applied to continue living in the area by September 30 had their application rejected.
Applicants can challenge a negative EU Settlement Scheme application by launching an appeal.
But the3Million, which campaigns for EU citizens' rights, is concerned about the status of those who are left "in limbo" waiting for their appeals to be concluded.
The EU Settlement scheme launched in March 2019 to regulate the immigration status of European citizens who live in the UK.
Those who have lived in the UK for five years, and meet the criteria, can receive settled status and remain in the country indefinitely.
Others who have lived in the country for less time can receive pre-settled status, which allows them to remain for a further five years. They can later apply for settled status.
The figures show that since applications opened, 2,080 people applied to continue living in Chesterfield, with 1,970 receiving a conclusion by the end of September.
Of them, 1,190 (60%) received settled status and 670 (34%) pre-settled.
The highest number of applications came from citizens of Poland (560), Romania (410) and Italy (180).
Though the scheme officially closed on June 30, EU citizens with limited reasonable grounds for missing the deadline can still apply to secure their rights.
Around 40 applications were submitted after the deadline in Chesterfield.
The Home Office said people with a pending application, are protected while the outcome of their application is unknown.
A spokeswoman said the EU Settlement Scheme has been an "overwhelming success", with 6.3 million applications received and 5.5 million people being granted permission to stay so far.
She added: “Caseworkers will always look for reasons to grant rather than refuse.
"Individuals can be refused on eligibility or criminality grounds, and if a refused applicant disagrees with our decision, they can apply for an administrative review or appeal.
“We have published non-exhaustive guidance on reasonable grounds for making a late EUSS application and take a flexible and pragmatic approach to considering them, and we’ve made millions of pounds available in funding for organisations to support vulnerable applicants.”