A bioactive disubstituted nonenolide, named truncatenolide, was produced by Colletotrichum truncatum, which was collected from infected tissues of soybean showing anthracnose symptoms in Argentina. This is a devastating disease that drastically reduces the yield of soybean production in the world. The fungus also produced a new trisubstituted oct-2-en-4-one, named truncatenone, and the well-known tyrosol and N-acetyltyramine. Truncatenolide and truncatenone were characterized by spectroscopic (essentially one-dimensional (1D) and two-dimensional (2D) ¹H and ¹³C NMR and HR ESIMS) and chemical methods as (5E,7R,10R)-7-hydroxy-10-methyl-3,4,7,8,9,10-hexahydro-2H-oxecin-2-one and (Z)-6-hydroxy-3,5-dimethyloct-2-en-4-one, respectively. The geometry of the double bond of truncatenolide was assigned by the value of olefinic proton coupling constant and that of truncatenone by the correlation observed in the corresponding NOESY spectrum. The relative configuration of each stereogenic center was assigned with the help of ¹³C chemical shift and ¹H–¹H scalar coupling DFT calculations, while the absolute configuration assignment of truncatenolide was performed by electronic circular dichroism (ECD). When tested on soybean seeds, truncatenolide showed the strongest phytotoxic activity. Tyrosol and N-acetyltyramine also showed phytotoxicity to a lesser extent, while truncatenone weakly stimulated the growth of the seed root in comparison to the control. When assayed against Macrophomina phaseolina and Cercospora nicotianae, other severe pathogens of soybean, truncatenolide showed significant activity against M. phaseolina and total inhibition of C. nicotianae. Thus, some other fungal nonenolides and their derivatives were assayed for their antifungal activity against both fungi in comparison with truncatenolide. Pinolidoxin showed to a less extent antifungal activity against both fungi, while modiolide A selectively and totally inhibited only the growth of C. nicotianae. The SAR results and the potential of truncatenolide, modiolide A, and pinolidoxin as biofungicides were also discussed.