Ancient trees, which are vital for biodiversity and cultural heritage, maintain species richness, ecosystem resilience, and societal interconnectedness. However, escalating global climate change and human activities have led to widespread declines in ancient tree diversity and habitat degradation. Hence, understanding ancient tree dynamics is vital for precise conservation strategies, fostering harmony between society and ecology. This study addresses the gap in understanding the factors influencing ancient tree biomass and diversity in human-dominated areas. This study first collected biomass equations for ancient tree species. The biomass of ancient trees within human-influenced regions of Guangdong Province was subsequently computed in detail. Finally, by employing a structural equation model, we sought to quantitatively elucidate the influence of natural variables (encompassing geography, topography, soil composition, and climatic conditions) alongside anthropogenic variables (including population density, per capita GDP, the human footprint, ancient villages, and religious sites) on both the biomass and species richness of ancient trees. The species with the highest total biomass included Ficus microcarpa L.f. (2.45 × 105 t), Ficus concinna (Miq.) Miq. (4.07 × 104 t), and Litchi chinensis Sonn. (3.16 × 104 t). Moreover, the biomass presented a pronounced phylogenetic signal and evolutionary conservation. According to the results of the structural equation model, across the entire study area, soil characteristics emerged as the most influential predictor among the natural factors (β = −0.14), whereas population density stood out as the foremost predictor among the anthropogenic factors (β = 0.23). Notably, both climatic conditions and anthropogenic factors contributed positively to an increase in ancient tree biomass (β = 0.02) (β = 0.07), albeit concurrently leading to a decrease in species richness (β = −0.05) (β = −0.08). In regions characterized by high levels of disturbance, anthropogenic factors indirectly increase biomass by increasing the abundance of large-diameter ancient trees (β = 0.28) while concurrently decreasing species richness (β = −0.11). Conversely, in areas with low levels of disturbance, anthropogenic influences also indirectly increase biomass (β = 0.19) by fostering the growth of large-diameter ancient trees while simultaneously promoting species richness (β = 0.01). This study investigated biomass and diversity factors in ancient trees within human-dominated regions of Guangdong Province. This study provides insights into ecosystem determinants, informing conservation strategies and contributing to biodiversity preservation amid societal and environmental dynamics.