Alien tree plantations are expanding globally with potential negative effects for native biodiversity. We investigated plant species diversity and composition in a Pinus radiata landscape in south-central Chile, a biodiversity hotspot, by sampling understory vegetation in different plantation age classes, along forest roads and in natural forest remnants in order to find effective conservation measures for native biodiversity. Plantations, including different age classes and roadsides, maintained high native species richness at the landscape scale but supported a completely different community composition than natural forests. Thus, natural forest remnants must be conserved as plantations cannot replace them. Certain natural forest species occurred frequently in mature plantations and can represent starting points for retaining natural elements in plantations. Generalist native and alien species benefited from plantation management, mainly in young plantations and along roadsides. Stand maturation and a closed canopy, though, reduced alien species occurrences within plantations. Along roads, shade-tolerant aliens should be monitored and removed as they can potentially invade natural forests. Native species conservation in plantations requires a holistic approach of the full mosaic of land uses including the protection of remaining natural forests, alien species monitoring along roadsides and patches with continuous canopy cover to reduce pressure by alien species.