We explored the multiple-spatial-scale diversity patterns of canopy and understorey north-temperate mature tree trunk spiders following a hierarchically nested design. We investigated local dynamics and dispersal limitations of canopy and understorey spider assemblages by using a combination of additive partitioning of richness and changes in species composition through spatially nested multivariate ANOVA. Community composition differed significantly between canopy and understorey assemblages and also at stand and site spatial scales, while beta richness was higher than expected from random at the site spatial scale only. Alpha richness did not differ from random at any spatial scale considered in the regional context of our study (tree, stand, and site) and was similar in the canopy and the understorey when rarefied. There was a shift in the contribution of alpha and beta richness as the spatial resolution increased because beta diversity contributed more to tree spider richness and alpha diversity to our site-level spider richness. Our study demonstrates that diversity patterns of mature tree trunk spiders in north-temperate hardwood forests are scale dependent in vertical and horizontal space: at our tree and stand spatial scales, local dynamics such as niche availability and species interactions may drive bark spider assemblage diversity since mean richness did not change significantly whereas species composition did.