Coastal vegetated ecosystems play an important role in carbon cycling and bacterial communities inhabiting coastal sediments are responsible for the remineralization and processing of organic carbon (OC). We collected 1 m-long sediment cores in Posidonia seagrass meadows from coastal and estuarine sites in Australia that differed in their sedimentary organic and inorganic carbon, nitrogen and mud contents. The metabolic diversity of sediment heterotrophic bacterial communities was characterized at different sediment depths, based on the utilization pattern of 31 individual carbon substrates using Biolog EcoPlatesTM. High metabolic diversity was recorded at both sites, but the carbon substrate utilization rates and the use of carbohydrates were higher at the coastal site compared to the estuarine site. The heterotrophic bacterial community in the coastal sediment appeared to metabolize a more diverse OC pool compared to the estuarine site, which might partly explain the differences in OC storage among the seagrass habitats studied. The Biolog EcoPlatesTM provided a useful tool for characterising the sediment heterotrophic bacterial communities in the meadows and sediment characteristics and biochemical composition of the organic matter played an important role in shaping heterotrophic bacterial communities and their carbon utilization rates, potentially affecting carbon accumulation and preservation within seagrass sediments.