Burrowing macrofauna is known to influence the diffusion properties and solute transport within sediments as well as promoting aerobic microbial processes. It has recently been argued that changes in diffusion scales also affect anaerobic microbial processes in sediments. We tested this contention on coastal sandy sediment using a widely employed sediment plug flux-incubation technique. Our results confirm that volume specific microbial carbon and nitrogen mineralization apparently are enhanced (6–15 and 3–9 times, respectively) in thin (1 mm) compared with thick (10 cm) sediment plugs incubated in anoxic seawater. However, differential accumulation of inhibiting substances among plug sizes cannot explain this phenomenon, as the same pattern was observed for plugs incubated in anoxic porewater. Instead, we found that microbial activity in the overlying water and biofilms along the walls of incubation chambers affects the results significantly. A low, but constant production of 3–7 (TCO2) and 0.5 (NH4+) nmol cm−3 d−1 in the water phase and 51–140 (TCO2) and 4–16 (NH4+) nmol cm−2 d−1in biofilms irrespective of plug thickness can explain a large part of the apparent diffusion scale dependent change in volume specific solute production in the sediment. Model considerations show that sediment independent water phase and biofilm reactions occurring in the incubation chambers result in highly overestimated volume specific rates for thin plugs. The overestimate is negligible for plugs thicker than 1 cm because the total reactions occurring within these larger sediment plugs exceed those in the water and biofilms considerably. We therefore conclude that most of the apparent inverse relationship between diffusion scales and anaerobic microbial processes in sandy sediment found by the use of flux-incubation chambers is an experimental artifact rather than a true diffusion scale dependent effect.