Landscapes located in the periphery of economic development, such as in parts of the Carpathian ecoregion, host remnants of both near-natural ecosystems and traditional agricultural land use systems. Such landscapes are important both for in situ conservation of natural and cultural biodiversity, and as references for biodiversity restoration elsewhere in Europe. This paper first reviews the contemporary understanding of benchmarks for biodiversity conservation in terms of ecosystems with natural disturbance regimes and pre-industrial cultural landscapes. Second, after providing a historical background, we review the challenges to natural and cultural biodiversity conservation and discuss current development trajectories. Third, we provide concrete examples from six Carpathian areas with different proportions of natural and cultural biodiversity. Fourth, we discuss the need for a diversity of management systems toward protection, management and restoration, spatial planning, and multi-sector governance for conservation of natural and cultural landscapes’ biodiversity. Finally, we stress the need to encourage integration of management, planning and governance of social and ecological systems to maintain natural and cultural biodiversity. The natural vegetation of the Carpathian Mountains is mostly forests and woodlands. Natural disturbances as wind, snow, frost, fire and flooding as well as insects and fungi resulted in forests characterized by old and large trees, diverse horizontal and vertical structures, and large amounts of dead wood in various stages of decay. While some near-natural forests remain, in most of the Carpathian ecoregion pre-industrial cultural landscapes evolved. Human use created traditional village system with infield houses, gardens, fields, meadows and outfield meadows and pastures, and woodlands which not only provide ecosystem services but also represent cultural heritage. The maintenance of natural and cultural biodiversity may require active management of species, habitats and processes. However, designing management systems that emulate natural and cultural landscape’s disturbance regimes is a major challenge requiring collaboration of private, public and civic sector stakeholders, and integration of social and ecological systems. Maintaining and restoring the traditional village system’s social capital as well as functional networks of protected areas and implementing sustainable forest management in managed forests are thus crucial. The Carpathian ecoregion forms a quasi-experiment with new country borders that have created stark contrasts among regions regarding natural and cultural biodiversity. This ecoregion can therefore be seen as a landscape-scale laboratory for systematic studies of interactions between ecological and social systems to support the development of an integrated landscape approach to biodiversity conservation and cultural heritage.