ContextThe loss of ancient forests threatens many species. Effective nature conservation needs information on how forest availability in the surrounding landscape in space and time determines the diversity of multiple taxa.ObjectivesWe explored the relationship between forest availability at different spatiotemporal scales and the diversity of various groups: vascular plants (woody species, ground layer), epiphytes (bryophytes and lichens), fungi (ectomycorrhizal, arbuscular mycorrhizal, pathogenic, saprotrophic), and carabid beetles. Besides the observed diversity, we also estimated dark diversity, i.e. suitable but absent species. Dark diversity is theoretically a sensitive metric in detecting ecosystem conditions as it is typically relatively large and contains susceptible species.MethodsWe recorded the observed diversity by field sampling and soil DNA in 100 temperate ancient old-growth forest sites in southern Estonia; dark diversity was estimated for the same sites using species co-occurrence data. Forest availability estimates were obtained from four topographic maps (1900s-2010s) at the 0.5–5 km radius.ResultsThe biodiversity of forest specialists was higher at larger historical forest availability at the spatial scale of 2–5 km radius. The diversity of light-demanding forest ecotone taxa mainly had negative relationships with young forests on previous agricultural lands (at 0.5–2 km radius). Dark diversity models were often more strongly associated with forest availability than observed diversity models.ConclusionsDark diversity enhances our understanding of how current and historical forest availability affects local biodiversity. As young forests cannot provide suitable habitats for many forest-dwelling species, stable forest habitats must be preserved as source areas to enhance biodiversity.