The oxygen dynamics of seagrass roots implicitly influences the associated microbial activity. The oxic zone surrounding roots creates a distinct electrochemical gradient where the concentration of oxygen is highest closest to the root tip and decreases sharply with distance away from the root. Microbes within each micro-niche utilise progressively less energy-favourable electron acceptors (e.g. oxygen, nitrate, sulphate). It is probable that oxygen-consuming bacteria would have greatest activity at or near the root tips of seagrass. This study assessed the potential of a community- level physiological profiling assay, based on a high throughput, microtitre-based oxygen sensor, to evaluate aerobic heterotrophs in seagrass ecosystems. Seagrasses were collected from shallow water within the Banana River Estuary, Kennedy Space Center. The sensitivity of the method for characterizing microbial communities associated with seagrasses was tested both spatially (i.e. rhizoplane, rhizosphere) and following imposed light stress (i.e. shading). Results showed that oxygen availability and competition for nutrients defined the microbial response to carbon substrates, with the fastest response and largest peaks for N-containing carbon sources (e.g. asparagine, natural extracts). Aerbic microbial activity was higher for Halodule wrightii than Syringodium filiforme. Primary nitrogen and secondary phosphorus limitation was observed for microbial activity from the rhizoplane and rhizosphere of H. wrightii. Shading appeared to reduce nutrient limitation for H. wrightii and reduced aerobic microbial activity. The microbial activity and functional response varied between the rhizosphere and rhizoplane, which suggests that microbial activity on this fine spatial scale needs to be considered when evaluating biogeochemistry associated with seagrass sediments.