The flow regime of the MacKenzie River, western Victoria, Australia, has been substantially modified since the construction of a water supply reservoir on its upper reach in 1887. Water is regulated at several locations downstream of the reservoir, creating a substantially modified flow regime, impacting key environmental values of the river. The river receives an environmental flow allocation and the river channel is used to transfer water dedicated for consumptive use. Ecological response models were developed using freshwater algal assemblages to assess the water quality and ecological condition of this working river under base flow and before and after freshes that deliver water to users. To assess the impact of the different flow regimes on river condition, ten sites were repeatedly sampled along the river between February 2012 and November 2014. Physical and chemical characteristics of water were measured as well as biological properties comprising the algal periphyton communities, including dry mass, ash-free dry mass, chlorophyll-a concentration and species composition. The lower reaches of the river were shown to be in poor condition under low flows, but this condition improved under flows of 35 ML/day, as indicated by the reduction in green algae and cyanobacteria and an improvement in DSIAR scores. The results will be used to tailor the duration and discharge of freshes used to deliver consumptive water to improve the condition of the stream thereby supplementing the flows dedicated to environmental outcomes.