The present study aimed to examine the capability of Clonostachys rosea isolates as a biological control agent against grapevine trunk diseases pathogens. Five C. rosea and 174 pathogenic fungal strains were isolated from grafted grapevines and subjected to in vitro confrontation tests. Efficient antagonism was observed against Eutypa lata and Phaeomoniella chlamydospora while mycoparasitism was observed to the pathogens of Botryosphaeria dothidea and Diaporthe spp. pathogens in in vitro dual culture assays. The conidia production of the C. rosea isolates were also measured on PDA plates. One isolate (19B/1) with high antagonistic capabilities and efficient conidia production was selected for in planta confrontation tests by mixing its conidia with the soil of Cabernet sauvignon grapevine cuttings artificially infected with B. dothidea, E. lata and P. chlamydospora. The length and/or the incidence of necrotic lesions caused by E. lata and P. chlamydospora at the inoculation point were significantly decreased after a three months incubation in the greenhouse on cuttings planted in soils inoculated with the conidia of strain 19B/1, while symptom incidence and severity were unaffected in the case of the pathogen B. dothidea. Based on the above results, we consider C. rosea a promising biological control agent against some grapevine trunk diseases.