The effect of burrowing activities of ghost shrimp (Neocallichirus limosus and Biffarius arenosus) and heart urchin (Echinocardium cordatum) on the flux of dissolved substances across the water–sediment interface was examined by comparing the transport of the tracer deuterium oxide (D2O) between sediments and overlying water in experimental laboratory tanks with and without fauna. This experiment was complemented by measurements of the diffusive flux of naturally occurring isotopes of radium (224Ra and 223Ra) from the sediments into the water column. Despite different burrowing habits, all three species enhanced flux of D2O and the Ra isotopes by similar amounts. In sandy sediments, both shrimps and urchins enhanced flux of D2O across the water–sediment interface 2.5 fold. In muddy sediments, however, results were inconclusive. There was little difference in flux of 224Ra and 223Ra between muddy and sandy sediments; flux was 2.3–3.9 times higher in the presence of the burrowing species than in their absence. These findings emphasize (i) the important role of burrowing macrofauna in the transport of dissolved substances between the sediment and water bodies and (ii) the dependence of various biogeochemical processes, including nutrient release, on the benthic macrofauna of Port Phillip Bay.