Background: Grapevine trunk diseases (GTDs) are a complex group of diseases that lead to major economic losses in all wine-producing countries. The investigation of biocontrol agents (BCAs) capable of forestalling or at least minimizing the development of GTDs has, recently, become a priority. Nursery experiments were set up to (i) assess the biocontrol effect of Trichoderma atroviride (Ta) SC1 and Bacillus subtilis (Bs) PTA-271, alone and in simultaneous application, against Botryosphaeria dieback (BOT)- and black-foot (BF)- associated pathogens during the grapevine propagation process and (ii) evaluate the success of the BCA inoculation during the grapevine propagation process, using quantitative reverse-transcription polymerase chain reaction techniques. RESULTS: The results demonstrated a significant reduction in the percentage of potentially infected plants and the percentage of fungal isolation from wood fragments of BOT and BF pathogens in nursery material treated with Ta SC1 and Bs PTA-271, respectively. In one of the experiments, simultaneous treatments with Bs PTA-271 and Ta SC1 caused a reduction in percentages of potentially infected plants and fungal isolation, from wood fragments containing BOT and BF pathogens. CONCLUSION: These biological treatments may be relevant components of an integrated approach, using complementary management strategies to limit infection by GTD pathogens, but further research is still needed to elucidate the effectiveness of Bs PTA-271 and the benefits of simultaneous application with Ta SC1 for the control of GTD pathogens in nurseries. (c) 2022 Society of Chemical Industry.