| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 2 | Holy Names |
A | Lugo | Valdes | Mulrooney | Derbes | 52.20 | FHSAA 2A District 10 |
| 3 | Tampa Catholic |
A | Greaves | Chinea | Green | Ritchie | 49.29 | FHSAA 2A Region 3 |
| 4 | Tampa Catholic |
B | Olivera | Velasco | Valladares | Pineda | NT | |
| 5 | Jefferson |
A | Bradford | Ellis-Williams | Grubbs | Nunn | 53.39 | FHSAA 2A Region 3 |