Rhodoliths provide numerous benefits to coastal ecosystems and help support high biodiversity. No study, however, has explored rhodoliths that occupy northeastern Gulf of Mexico patch reefs, and their contributions to local ecosystem function remain uncharacterized. Here, we employed the acetylene reduction assay to assess nitrogen fixation capability in rhodolith holobionts (Lithothamnion spp.; Rhodophyta), sediment, and surrounding seawater from a subtropical patch reef ecosystem in the northeastern Gulf of Mexico. We found no evidence for nitrogenase activity in rhodolith holobionts or seawater from our study site, while nitrogenase activity in sediment underlying rhodoliths was approximately equivalent to a nitrogen fixation rate of 0.521 (SD 0.087) nmol N2 g dry mass−1 hr−1. Our results suggest that rhodoliths in the northeastern Gulf of Mexico rely on sources of nitrogen from sediment nitrogen fixation or water column nutrient availability rather than the activity of symbiotic diazotrophic microorganisms. Functional analyses recognizing rhodoliths as holobionts warrant further investigation to better understand the ecology of rhodoliths.