The activity of six extracellular enzymes involved in the degradation of dissolved organic carbon compounds was measured in two highly urbanised and two minimally impacted streams east of Melbourne, Australia, using 4-methylumbelliferyl-substrates. Small-scale temporal variation in enzyme activity was determined by repeatedly sampling the same point in the water column, while the effect of flow was determined by sampling in regions of higher and lower flow in both stream types. Replicate samples showed that enzyme activity was not significantly different over small (minutes) time scales. On five of six sampling occasions the enzyme activity was unaffected by flow. On one sampling occasion in a minimally disturbed stream, the difference between the high- and low-flow regions was statistically significant (ANOSIM, Global R = 0.78, P = 0.03). Enzyme activity profiles (activities of the suite of enzymes) of the streams in urbanised catchments were different to those in minimally disturbed catchments. The measurements made in four different streams showed high reproducibility over short time periods (minutes) which lends greater credibility to analogous spatial studies. Although these results determined that small-scale temporal variability was not significant, and that the effects of flow were generally minimal, it is recommended that spatial and temporal variability in the stream be at least considered before any studies measuring extracellular enzyme activity in stream waters are carried out. Such an approach will lead to conclusions from measurements that are not likely to be confounded by variables such as flow rate or time.