James Anthony Battista has been charged with rape of a child, unlawful contact with a minor, aggravated indecent assault, indecent assault and endangering the welfare of children.
Battista ran Little Friends Day Care in Penn Township, along with his wife.
The day care has since closed.
Last June, Pennsylvania State Police Avondale Barracks received a report that Battista had sexually assaulted a child who attended the day care when the child was 3 years old.
During the course of the investigation, three other children were identified as victims. These children would have ranged in age from 3 to 7 years old at the time of the alleged assaults, which spanned from 2006-2009.
Pennsylvania State Police said they fear there are more victims who have yet to come forward.
Battista was taken into custody Wednesday.
He was arraigned and remanded to Chester County Prison on $500,000 straight bail pending his preliminary hearing scheduled for next week.
Anyone with information on this investigation is asked to contact Tpr. Stefano Gallina at the Avondale Barracks at 610-268-2022.