The Niobrara Valley supports exceptional biological diversity within its narrow confines, where elements of the following ecosystems exist in the same area or very close to each other: northern boreal forest, ponderosa pine forest and savanna, eastern deciduous forest, tallgrass prairie, mixed-grass prairie, and sandhills prairie. Approximately 160 species of plants and animals found in the Niobrara Valley are at the edge of their range. Some plant and animal species are state or federally listed as rare, threatened, endangered, or candidate species.
The braided lower river provides important nesting habitat for the endangered interior least tern and threatened piping plover. The river also provides important migratory habitat for endangered whooping cranes, bald eagles, and the recently delisted peregrine falcon. Side tributaries or "spring branch canyons" provide essential habitat to the endemic Bailey's eastern woodrat and the olive-backed pocket mouse. The side tributaries also provide important habitat to several glacial relict fish species including pearl dace, northern redbelly dace, and blacknose shiners.
In addition to these biologically significant vertebrate species unique to the Niobrara Valley, invertebrate species also have a special niche. Some 92 species of butterflies have been recorded in the Niobrara Valley and sixteen species reach the edge of their range here. Hybridization of three species, Red-spotted purple, Weidemeyeri's admiral, and Eastern viceroy are noted as evolutionary and genetically significant.