| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 3 | Oak Ridge |
A | Clarke | Oracius | Suffrant | Altidor | NT | |
| 4 | West Orange |
A | Brown | Carson | Glover | Dillard | 42.96 | 42.96 West Orange - Dr. Baptiste Invitational 2024 |
| 5 | Edgewater |
A | Senatus | Sterling | Lewis | Jenkis-Lee | 41.55 | FHSAA 3A Region 2 |
| 6 | Winter Park |
A | Diaz | Sutton | Jackson | Knight | 42.47 | 42.47 Charlie Harris Relays |
| 7 | University (Orlando) |
A | Baker | Grullon | McGinnis | Moyer | DNS | FHSAA 4A Region 1 |