Ink disease is a devastating disease of chestnut (Castanea sativa) worldwide, caused by Phytophthora species. The only management measures of this disease are chemical and agronomic interventions. This work focuses on the evaluation of the in vitro antagonistic capacity of 20 isolates of Trichoderma spp. selected in a diseased chestnut orchard in Tuscan Apennines (San Godenzo, Italy) for the biocontrol of Phytophthora xcambivora. Each Trichoderma isolate was tested to investigate pathogen inhibition capability by antagonism in dual cultures and antibiosis by secondary metabolites production (diffusible and Volatile Organic Compounds). The six most performing isolates of Trichoderma spp. were further assessed for their aptitude to synthesize chitinase, glucanase and cellulase, and to act as mycoparasite. All six selected isolates displayed the capability to control the pathogen in vitro by synergistically coupling antibiosis and mycoparasitism at different levels regardless of the species they belong to, but rather, in relation to specific features of the single genotypes. In particular, T. hamatum SG18 and T. koningiopsis SG6 displayed the most promising results in pathogen inhibition, thus further investigations are needed to confirm their in vivo efficacy.