1. Bacterial extracellular enzymes provide a measure of microbial response to organic matter supply, pivotal to the recovery of riverine food webs after disturbances such as floods. 2. We examined the effects of flood duration on extracellular enzyme response from riverbank and floodplain wetland sediments from the Murrumbidgee River, south-east Australia. 3. There were strong temporal peaks in enzyme activity from both riverbank and billabong sites, peaking between 1 and 5 days following flooding and with a general decline by 21 days. A dominance of non-glucosidase and xylosidase enzymes resulted in no significant differences between billabong and riverbank sediments. This supports the hypothesis that regulated Australian river systems are driven by autochthonous carbon sources. 4. The short response time of the glucosidase after flooding suggests that even short pulses (24 h) in high flows may stimulate bacterial activity, as dissolved organic carbon (DOC) loads also peak at this time. However, a longer wetting time may be needed to drive hydrolysis of the proteins, fatty acids and longer chain polysaccharides, whether in the littoral zone of the river or connection with the floodplain.