Chestnut trees (Castanea sativa) are important forest and agricultural plants in northern and central Portugal, holding significant ecological, economic, and cultural value. However, chestnut ecosystems are increasingly threatened by a combination of devastating diseases and the growing impacts of climate change. Pathogens such as Phytophthora cinnamomi, responsible for ink disease, Cryphonectria parasitica, the causal agent of chestnut blight, and the more recently emerged Sirococcus (=Gnomoniopsis) smithogilvyi, associated with brown rot of nuts, pose severe and compounding threats to tree health and productivity. These biotic stresses are further intensified by abiotic pressures, particularly drought and extreme temperature events, which weaken tree defenses and create conditions more favorable to pathogen spread. Conventional disease management strategies, largely reliant on chemical inputs, are increasingly recognized as insufficient and environmentally unsustainable, underscoring the urgent need for innovative, nature-based approaches that can simultaneously address disease pressure, promote plant vigor, and enhance resilience to environmental stress. In this context, we sought to develop microbial formulations with biostimulant properties that promote chestnut phytosanitary health and climate resilience, reduce dependence on chemical inputs, and advance a regenerative agricultural model. Microorganisms were isolated from healthy and declining chestnut ecosystems, sampled across reproductive and vegetative chestnut tissues and rhizospheric soil to capture the microbial diversity naturally associated with these environments. A preliminary screening evaluated the biotechnological potential of the recovered isolates. Antagonistic activity against the three pathogens was assessed in vitro using dual-culture assays, with several isolates showing inhibitory effects against one or more pathogens, indicating promising biocontrol potential. Isolates were also screened for plant growth-promoting traits: phosphate solubilization was assessed on Pikovskaya medium, and siderophore production using Chrome Azurol S agar. Multiple isolates tested positive for both traits, suggesting roles in nutrient mobilization, iron acquisition, and enhanced stress tolerance. To further characterise associated microbial communities, bacterial and fungal diversity in the rhizospheric soil is being assessed through 16S rRNA and ITS amplicon sequencing, complemented by qPCR quantification of key pathogens, providing both culture-based and community-level perspectives on the chestnut microbiome. These results revealed a diverse and potentially valuable microbial community naturally associated with chestnut, with promising applications in sustainable disease management and climate adaptation. The most encouraging isolates will be characterised at the molecular and functional level and integrated into microbial consortia, with a view to developing bio-based formulations improving chestnut phytosanitary status and resilience to climate stress, contributing to a more sustainable model of chestnut forest management.