We contributed to the incorporation of spatially explicit, quantitative methods in Melbourne’s Healthy Waterway Strategy, and participated in stakeholder deliberations that used the results to identify actions and targets for ecological values in the strategy’s participatory framework. We developed habitat suitability models for 52 macroinvertebrate families, 13 native fish species, and platypus, and applied quantitative methods (Zonation) to prioritise cost-effective management actions throughout the >8,000-km stream network to optimise protection and restoration of instream animal diversity. The benefits of this approach (that were not possible with previous approaches to prioritizing investment) included: better use of available biological data, with discrete, point-location data used to generate spatially continuous estimates of instream biodiversity in unsurveyed reaches; improved granularity in mapping of biodiversity patterns, alerting stakeholders to unrecognised values, constraints and opportunities; ability to integrate and model strategic considerations such as different aspects of climate change impacts (warming, drying), land use change and their interactive effects; ability to quantify the expected difference made by management actions, and to account for costs so that action planning can be based on cost-effectiveness; ability to spatially prioritise management actions, and to interrogate and critically debate alternative actions at specific locations for planning and target setting; improved ability to map, summarise and communicate decision-relevant data to different audiences; and repeatable analyses that can be scrutinised, error-checked, critiqued and built upon. The quantitative tools required substantial communication in the diverse participatory settings of the strategy, but were well received, and acknowledged as a significant advance on past approaches.