In response to the need to augment electrofishing surveys of golden perch, Macquaria ambigua (an important endemic freshwater fish in southeastern Australia), with other non-lethal survey methods, the utility of environmental DNA (eDNA) as an index of relative fish abundance/biomass was investigated. From three adjacent rivers, five sites in each were sampled for eDNA immediately before electrofishing during sequential years. Up to six individuals or 10 kg of electrofished golden perch (200–548 mm total length) were caught or observed site−1. Analyses of concurrent eDNA concentrations revealed no significant relationship with the relative abundance of golden perch, but there was with relative biomass—manifesting as increasing eDNA concentrations between 1.1 and approximately 5.5 kg biomass site−1, after which concentrations stabilized at greater biomasses. Future research warrants assessing the viability of sampling eDNA for spatio-temporally monitoring rivers where low biomasses of golden perch (1–5 kg site−1) are likely to occur.