The need for accurate monitoring data to support effective marine management is ever more pressing under increasingly uncertain climate conditions. The diversity and scale of Australia’s marine environment poses considerable challenges to develop effective monitor strategies. Novel methods such as environmental DNA (eDNA) surveys can provide tree-of-life community data cheaper and on larger scales than many established methods. The processing and analysis of eDNA samples is usually outsourced to external service providers, yet questions remain around comparability of outcomes and how standardisation might affect results. We collected eDNA samples from rocky reefs and soft sediment habitats in tropical (Rowley Shoals, WA) and temperate (Hobart, TAS) regions, and analysed them in five different laboratories. Laboratories started with the same samples and used both a standardised and lab-specific workflow. The results of all laboratories showed high similarity at broad ecological scales (region, habitat, site). However, more variability was present in regions with high biodiversity levels and when lab-specific workflows were more divergent. Results of this study suggest that workflow standardisation is advisable to improve comparability and that increased replication levels are necessary in regions with high biodiversity.