Investigators say Andrew C. Glidwell assaulted the child Friday at the Children First Preschool at the First United Methodist Church in Hendersonville, the Gannett Tennessee news service reported.
Bail was set at $200,000. Glidwell is scheduled to appear in court on May 27.
Hendersonville Police Chief Terry Frizzell said detectives will be talking to other children at the school.
"We have to assume there are potentially other victims," he said.
State authorities say that the preschool can enroll up to 160 children from babies up to 5 years old.
Michael Williams, the First United pastor, said the church is cooperating with investigators.