Dehesa is a unique ecosystem associated with high biodiversity, that integrates trees, livestock, and pasture, making agro-pastoral production compatible with sustainability. However, in the last few decades, a manifold of factors have caused a decline in tree vitality, density, and coverage, leading to long-term changes in species composition and ecosystem structure. This study aims to determine changes in the diversity of the herbaceous plant community in relation to environmental characteristics, the phytosanitary state of the holm oak (Quercus ilex L. subsp. ballota (Desf.) Samp.), and possible interactions with biotic agents, including Phytophthora cinnamomi. For this purpose, the floristic composition and alpha diversity of the understory were assessed in two dehesa stands in Southern Spain. Additionally, the spatial heterogeneity (beta diversity) patterns of herbaceous plants were evaluated in relation to a climatic gradient and subplot orientation at the regional, plot, tree, and subplot scales. Our findings show that microsite features and climate substantially impact the herbaceous plant community in dehesa stands. Annual precipitation is a crucial factor affecting the diversity and biomass of herbaceous plants on a broader, regional scale, consistent with its role as a limiting factor in the Mediterranean climate. However, site-level differences, such as soil clay content and plot slope angle, positively correlate with plant biodiversity, growth, and richness, varying with the Biodiversity Index considered. Moreover, microsites resulting from the combined effects of plot and tree are the main drivers of dissimilarities between samples, as expressed by beta diversity. Contrary to our initial hypothesis, our results reveal no significant association between tree health and herbaceous biodiversity decline.