| La Petite Academy of Middleburg | 3150 County Road 220, Middleburg, FL 32068 | Preschool |
| Annunciation Catholic School | 1610 Blanding Blvd., Middleburg, FL 32068 | Preschool |
| Backpack University | 400 College Drive, Middleburg, FL 32068 | Center |
| Doctors Inlet Elementary Preschool | 2634 Cr 220, Middleburg, FL 32068 | Preschool |
| Episcopal Children's Services Early Learning Center | 2506 Blanding Boulevard, Middleburg, FL 32068 | Preschool |
| Fox Meadows Chrisitian Daycare | 3227 Old Jennings Road, Middleburg, FL 32068 | Preschool |
| Good Samaritan Learning Tree | 3813 Old Jennings Road, Middleburg, FL 32068 | Preschool |
| Kid's World Child Center | 5390 County Road 218 West, Middleburg, FL 32068 | Preschool |
| KidsFirst Learning Center | 2645 Blanding Blvd, Middleburg, FL 32068 | Preschool |
| O.P.K. South of Middleburg | 3425 Habitat Drive, Middleburg, FL 32068 | Center |
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| Pinewood Christian Academy | 198 Knight Boxx Rd, Middleburg, FL 32068 | Preschool |
| Rideout Elementary Preschool | 3065 Apalachicola Blvd, Middleburg, FL 32068 | Preschool |
| Tender Hearts Child Care | 29 Cinnamon Street, Middleburg, FL 32068 | Center |
| Tynes Elementary Preschool | 1550 Tynes Blvd, Middleburg, FL 32068 | Preschool |
| Wee Care DayCare & Preschool, Inc | 4717 County Road 218, Middleburg, FL 32068 | Preschool |
| Wilkinson Elementary School Preschool | 4965 County Road 218, Middleburg, FL 32068 | Preschool |
| YMCA Pryme Time at Coppergate Elementary | 2250 County Road 209 North, Middleburg, FL 32068 | Center |
| YMCA Pryme Time at Drs. Inlet Elementary | 2634 State Road 220, Middleburg, FL 32068 | Center |
| YMCA Pryme Time at JL Wilkinson | 4965 County Highway 218, Middleburg, FL 32068 | Center |
| YMCA Pryme Time at Middleburg Elementary | 3958 Main Street, Middleburg, FL 32068 | Center |
| YMCA Pryme Time at Rideout Elementary | 3065 Apalachicola Boulevard, Middleburg, FL 32068 | Center |
| YMCA Pryme Time at Swimming Pen Creek | 1630 Woodpecker Lane, Middleburg, FL 32068 | Center |
| YMCA Pryme Time at Tynes Elementary | 1550 Tynes Boulevard, Middleburg, FL 32068 | Center |