Habitat loss is a primary threat to seahorses, pipefishes, and their relatives (family Syngnathidae) globally. Conservation intervention is difficult or ineffective when species lack critical habitat information. To better understand the habitat associations of syngnathids we conducted underwater visual surveys of 79 sites in three habitat categories (Reef, Continuous, and Discontinuous Submerged Rooted Vegetation) in Biscayne National Park (BNP), a coastal Marine Protected Area (MPA) in southeastern Florida. We recorded nine syngnathid species in our surveys; presence/ absence data was analyzed alongside a suite of abiotic and biotic variables. Nonparametric statistical tests indicated that syngnathids were significantly more abundant inside Biscayne Bay and we did not detect any syngnathids at reef sites—located outside Biscayne Bay—despite reef-associated species being previously reported in BNP. Additional analyses indicated that sites where syngnathids were present were characterized by fine sediment, reduced horizontal visibility, high seagrass coverage, and low coverage of sessile benthic invertebrates and reef-associated turf algae. The most important predictor of syngnathid presence was a predominance of fine sediment—an indicator of low-velocity hydrodynamic conditions—and seagrass coverage at occupied sites was nearly double that of unoccupied sites. Variation in habitat use among the three most abundant sympatric syngnathids (Hippocampus zosterae, S yngnathus scovelli, and Syngnathus floridae) indicates niche partitioning based on salinity, local hydrodynamic regime, and seagrass coverage. Our study points to the value of protecting sheltered seagrass beds; heightened vulnerability to habitat change—driven by watershed development, impacts from fisheries, recreation and tourism, and climate change—necessitates coordinated management of land use outside of the protected area.