| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 3 | Windermere |
B | Brown | Beck | Gil Rodriguez | Cameron | NT | |
| 4 | First Academy (Orlando) |
B | Awosika | Wilson | Spafford | Schmid | 51.03 |
| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 2 | First Academy (Orlando) |
A | Adams | Valois | Dickson | Lozada Da Silva | 52.04 | |
| 3 | University (Orlando) |
A | John | Kumah | Mendez | Ramirez | DNS | |
| 4 | Lake Nona |
A | Moss | Kimmons | Lee | Stephens | 50.00 | |
| 5 | Windermere |
A | Gordon | So | Mincey | Robinson | 50.92 | |
| 6 | Colonial |
A | Sukut | Smith | Arroyo | Rodriguez | 51.05 | FHSAA 4A Region 1 |
| 7 | Tohopekaliga |
A | Pagan | Reid | Francois | Charles | 52.15 | Orange Belt Conference Championships |
| 8 | Innovation_Orlando | A | McNeill-Toro | Londoni | Batts | Turner | 53.50 | Harmony Invitational |