The exploitation of Australia's fisheries, valued at over $2.5 billion annually, lack appropriate stock assessments for many species. This deficit is primarily due to the low economic value of many individual stocks, high monitoring costs, and reliance on low-quality fishery-dependent data. Developing cost-effective, fishery-independent population monitoring approaches is crucial. We designed a two-phased project to assess if environmental DNA (eDNA) methods could be used to estimate the relative abundances or biomasses of Murray cod, mulloway, golden perch and bony herring. Phase 1 comprised a definitive literature review of 63 studies and revealed that eDNA analysis presents a promising genetic method for assessing fish-population changes—justifying further research on Australian species. Phase two comprised manipulative experiments including aquaria trials to examine relationships between eDNA concentration and biomass, and field trials evaluated these associations under natural conditions. Positive relationships were found for all species, although these were strongly influenced by various factors such as water flow and temperature and season. The results support continued collection of eDNA samples alongside conventional sampling to refine the relationships between fish eDNA concentrations and biomass. Environmental DNA methods are advancing towards becoming a realistic option for estimating the relative biomass of Australian species in the future.