Botryosphaeria dieback is a widespread grapevine trunk disease caused by species of fungi within the family Botryosphaeriaceae. Symptoms of the disease include bud necrosis, leaf chlorosis, vascular discoloration of the wood, cankers and dieback. In 2013 typical symptoms of Botryosphaeria dieback was observed in Western Sicily and four species associated with the syndrome were isolated and identified as Diplodia seriata, Lasiodiplodia mediterranea, Neofusicoccum parvum and Neofusicoccum vitifusiforme. In order to fulfill Koch’s postulates, pathogenicity tests of eighteen isolates were performed on two-year grapevine cuttings of cv. Inzolia. Six months after inoculation all the Botryosphaeriaceae isolates induced vascular discoloration of the wood with differences between the species and within the isolates. D. seriata B22 was the most virulent isolate showing the longest mean lesion length, significantly different from those caused by any other Botryosphaeriaceae isolates. The control of Botryosphaeria dieback is difficult and integrate pest management is necessary to reduce disease incidence. In recent years, the use of biocontrol agents is becoming a valuable strategy for the control of plant diseases. Bacillus amyloliquefaciens AG1, isolated from grapevine wood tissue, has been reported to be effective against some fungi associates to grapevine trunk disease. In this study, the antimicrobial activity of B. amyloliquefaciens AG1 was evaluated by well diffusion assay against Botryosphaeriaceae fungi associated to Botryosphaeria dieback in Western Sicily. Cell-free supernatant of B. amyloliquefaciens AG1 showed significant antifungal activity inhibiting fungal growth of all tested isolates.