Neofusicoccum parvum is a wood-colonizing fungus capable of infecting numerous economically important crops. In particular, this fungal trunk pathogen represents a significant threat to worldwide grape production. Neofusicoccum parvum’s ability to colonize and damage the host may rely on the secretion of diverse effectors, such as phytotoxic specialized metabolites. Despite the scientific efforts to discover and understand the role of specialized metabolites in disease development, there is a lack of information on the factors influencing the phytotoxin production in N. parvum and how this pathogen modulates its metabolome when facing the host. In this study, we evaluated the specialized metabolites production of two N. parvum wild-type strains (Bt67 and NpB) and a UV mutant (UV9) cultured in 20-L bioreactors for six days. To give insight into how N. parvum adapts the specialized metabolite production in contact with host tissue, the three strains were cultured in the presence and absence of grapevine cane powder. Daily ethyl acetate extracts were analyzed using HPLC-MS/MS. This dataset corresponds to the mzML files generated from the culture filtrates of the three strains grown with or without grapevine cane powder.