The escalating impacts of climate change have heightened concerns about the frequency and severity of natural disasters, particularly extreme flooding events. Future projections underscore the necessity for innovative flood prevention strategies, including broad-scale nature-based solutions. Here, we present the first comprehensive assessment of the flood prevention benefits provided by Canadian natural ecosystems and identify key areas crucial for human well-being. Using spatially explicit modeling, we (1) evaluated the potential runoff retention by natural ecosystems and (2) identified downstream urban and agricultural areas critically dependent on these natural benefits, particularly those in floodplains and close proximity to upstream natural ecosystems. The natural ecosystems within the top 5 % of sub-basins, representing regions with a high priority for conservation practices aimed at flood prevention, play a crucial role in safeguarding approximately 54 % (∼6,000 km2) of the total built-up area and 74 % (∼16,900 km2) of the total cropland situated within floodplains. Additionally, they are positioned upstream of floodplain-based urban zones belonging to 358 population centers, directly benefiting 3.7 million people (∼10 % of the Canadian population) and indirectly benefiting almost 20.1 million people (∼56 % of the Canadian population). Moreover, among Canada’s 5.2 million km2 of flood-preventing natural ecosystems, we identified a small fraction (10 %) whose loss or degradation would result in a significant (>50 %) increase in runoff. Several of these crucial ecosystems are situated in less populated northern regions, where local governments might want to incentivize conservation initiatives to support flood prevention. Our research underscores the imperative to integrate nature-based solutions into national strategies that consider the results of spatial planning analyses. Establishing other effective area-based conservation measures in the priority regions highlighted in this study can contribute towards reaching current ambitious environmental goals and provide critical flood prevention benefits. Additionally, our methods are transferable to other regions worldwide, leveraging globally available datasets and ensuring computational feasibility.