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There is evidence that secondary metabolites are involved in the fungal pathogenicity and virulence of <i>Neofusicoccum</i> spp. Fatty acids may also influence the plant&#8315;pathogen interaction but, so far, no information is available on their production by species of <i>Neofusicoccum</i> associated with Botryosphaeria dieback, which is a well-known syndrome of several plants with a complex etiology. In the present paper, the production of fatty acids in liquid medium, by strains of <i>N. vitifusiforme</i> and <i>N. parvum</i> associated with declining Sicilian vine plants, was evaluated. Data, acquired via gas chromatography&#8315;mass spectrometry (GC/MS), show the presence of linoleic acid as the most abundant fatty acid produced by both examined strains. In addition, the pathogenicity of <i>N. vitifusiforme</i> was tested on 2-year-old grapevine plants of cv. Inzolia.