Nine strains of the fungus Phomopsis spp. were isolated from a vineyard showing decline from the disease esca. Strains were screened for their ability to produce secondary metabolites showing chemical diversity. The culture extracts of each strain were analyzed by liquid chromatography-ultraviolet-diode array detection-mass spectrometry. Three strains were selected for the isolation and characterization of eight of the major metabolites. Structures were elucidated by spectroscopic analyses including two-dimensional NMR and mass spectrometry and by comparison to literature data. Among the isolated metabolites were the known phomopsolide B (1), sydowinin A (6), sydowinol (7), cytosporone B (8), and four new furanones named phomopsolidones A-D (2-5). The fungal strains were identified as Phomopsis sp., Phomopsis viticola Sacc and, Phomopsis viticola complex. Biological assays on Vitis vinifera leaves and callus tissue, antibacterial, and insecticidal activities were evaluated. The results revealed variability regarding secondary metabolites with species of Phomopsis sp. associated with grapevine, raising the question of cultivar-driven strain selection and phytotoxins biosynthesis in grapevine plants.