| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 4 | Edgewater |
A | Barkett | Green | Hutchinson | Brown | 49.98 | FHSAA 3A Region 2 |
| 5 | Lake Brantley |
A | White | Oliver | Joseph | Robinson | 48.76 | FHSAA 4A Region 1 |
| 6 | Poinciana |
A | Duncan | Archelus | Raymond | Calderon | 49.46 | FHSAA 4A Region 2 |
| 7 | Winter Park |
A | Rodriguez | Leon | Hamilton | Eady | DNS |