Environmental flow rules are developed to provide a flow regime necessary to maintain healthy river and floodplain ecosystems in rivers regulated for human uses. However, few studies have experimentally assessed potential ecological mechanisms causing declines in the health and productivity of freshwater fish assemblages in regulated rivers to inform the development of appropriate environmental flows. We tested whether an experimental flow release in a regulated tributary of the Hunter River, Australia, altered the diet of two widely distributed fish species (Australian smelt Retropinna semoni and Cox's gudgeon Gobiomorphus coxii ) compared with data from unregulated reference and regulated control tributaries. Neither species had significant differences in the number of prey taxa ingested, gut fullness or composition of gut contents due to the environmental flow release (EFR). The diet of R. semoni did not differ significantly between regulated and unregulated tributaries in either catchment. However, the diet of G. coxii differed in only one of the two pairs of rivers consistently across all sample times. Assuming the EFR was sufficient to alter the composition of prey available for consumption by the fish species studied, our findings imply that functional indicators, such as the diet of generalist higher-order consumers, may be more suitable indicators of long-term flow regime change rather than short-term flow events.