The reinstatement of natural flow regimes is a rapidly emerging issue in river restoration worldwide. In northern Victoria, Australia, efforts are presently underway to restore a natural, intermittent flow regime to several streams which have received perennial diversions for both irrigation and stock and domestic water‐supplies for over 100 years. A pipeline to deliver water to landholders will significantly reduce transmission losses throughout the system allowing irrigation canals and diversion weirs to be decommissioned. The motivation for flow alteration in this system lies primarily in reducing inefficiencies in water delivery which, in turn, will be used to meet escalating demands on water resources. The ecological impact of the flow regime shift on these streams is likely to be substantial. This study utilized an existing artificial hydrological gradient (from perennial to intermittent) in two creek systems, to explore relationships between flow regime and a range of ecological variables. These data provide a benchmark against which to assess ecological changes once flow has been altered and form the basis for predicting changes that can assist future management decisions. Data collected from 10 sites across a strong hydrological gradient detected clear differences in geomorphology, water quality and biotic assemblages (macrophytes, macroinvertebrates and fish). By examining the relationship between flow regime and the distribution of biota we identify both the positive and negative outcomes of restoring naturally intermittent flow regimes within artificially perennial lowland streams. The reinstatement of intermittent flow regimes in artificially perennial streams will continue in many parts of the world as water delivery via these systems becomes increasingly uneconomical. While flow restoration may in principle be regarded as a positive step, these findings emphasize the need to consider fully the ecological consequences of restoring historical hydrological regimes to streams within the context of other human induced catchment disturbances.