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Abstract The Pantanal is a wetland located in the central region of South America (150,355 km2), mainly in Brazil (nearly 140,000 km2), touching Paraguay and Bolivia. The floodplain is fed by the Paraguay River and its tributaries, where the headwater of the rivers is located on the surrounding upland. Only 40% of this plateau retains its natural vegetation. Although the natural habitats of the Pantanal are still considered well preserved, retaining 80% of the vegetation cover, the biome faces environmental and socioeconomic threats that affect its biodiversity. The seasonal flux of the rivers is a fundamental ecological element for regional biodiversity. Habitat alteration and deforestation have affected terrestrial and aquatic habitats and their associated wildlife. This work identifies and analyses the threats to biodiversity considering land use, based on research experience in the region plus field work. Based on this, it discusses a strategic action plan for the biome's biodiversity.